Cell lines and culture conditions
The following commercially available cell lines were used: MCF10A (ATCC, CRL-10317); MCF10A with PIK3CAp.H1047R/+ knockin and matched PIK3CA+/+ control (PerkinElmer/Horizon Discovery, HD 101-011); MCF10A with AKT1E17K/+ (Perkin Elmer/Horizon Discovery, HD 101-007); SUM159 (Asterand Bioscience/BioIVT, SUM159PT); MDA-MB-468 (ATCC, HTB-132); T47D (ATCC, HTB-133); NIH-3T3 mouse embryonic fibroblasts (ATCC, CRL-1658); HEK293T (ATCC, CRL-11268). The MCF10A cell lines stably expressing doxycycline-inducible AKT2-E17K from the pTRIPZ vector were previously described37.
Maintenance culture conditions
All MCF10A-derived cell lines were maintained in standard MCF10A growth medium without antibiotics (DMEM/F12 medium (Wisent Bioproducts, 319-075-CL), 5% horse serum (Gemini Bio, 100508), 10 µg ml−1 insulin (Thermo Fisher Scientific/Gibco, A11382II), 0.5 mg ml−1 hydrocortisone (Sigma-Aldrich, H4001), 20 ng ml−1 EGF (R&D Systems, 236-EG-01M) and 100 ng ml−1 cholera toxin (List Biological Laboratories, 100B)). MCF10A stably expressing doxycycline-inducible HA-AKT2-WT and HA-AKT2-E17K (pTRIPZ constructs) were maintained in 0.5 μg ml−1 puromycin. NIH-3T3 mouse fibroblasts were grown in DMEM (Wisent Bioproducts, 319-005-CL) with 10% heat-inactivated FBS (Thermo Fisher Scientific, 10438026). SUM159, MDA-MB-468 and T47D cell lines were maintained in RPMI 1640 (Wisent Bioproducts, 350-000-CL) with 10% heat-inactivated FBS. Cell lines stably expressing PANK2 or PANK4 variant constructs in pLenti-Ubc-IRES-Neo (pLUIN) were maintained in 250 µg ml−1 G418 (Geneticin; neomycin analogue) (Invivogen, ant-gn-1). All of the cell lines were grown at 37 °C under 5% CO2 and high humidity, and were typically trypsinized and split every 2 days, keeping cell densities subconfluent. Cells were confirmed to be negative for mycoplasma contamination using the MycoAlert Detection Kit (Lonza, LT07-218).
Induction of HA–AKT2 with doxycycline
For experiments involving MCF10A stably expressing doxycycline-inducible HA–AKT2(WT) or HA–AKT2(E17K), cells were plated in standard MCF10A growth medium containing 200 ng ml−1 doxycycline (Sigma-Aldrich, D3447) for 30 h and then serum/growth factor deprived in medium containing 200 ng ml−1 doxycycline for 18 h for a total of 48 h. Doxycycline was not included in the medium on the day that cells were treated and collected.
RNA interference using siRNAs
MCF10A siRNA knockdowns
Stocks of siRNA were prepared by resuspending siRNAs in 1× siRNA buffer (5× siRNA buffer, (PerkinElmer/Horizon Discovery, B-002000-UB-100), diluted to 1× in sterile molecular-grade RNase-free water (PerkinElmer/Horizon Discovery, B-003000-WB-100)) for a stock concentration of 20 μM. After addition of buffer, stocks were vortexed for 30 min at room temperature, aliquoted into screwcap vials with gaskets, snap-frozen in liquid nitrogen and stored at −80 °C. Each aliquot was freeze–thawed no more than five times. For each target, three different validated siRNAs were pooled at equal concentrations such that the final total concentration in culture medium was 20 nM. For siRNA cell culture treatments in 6 cm dishes, siRNAs were diluted in 250 μl of prewarmed OptiMEM (Thermo Fisher Scientific/Gibco, 31985062) in tube A so that the final concentration on cells would be 20 nM total. At the same time, Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific/Invitrogen, 13778075) was diluted in 250 μl prewarmed OptiMEM in tube B so that the final concentration on cells would be 2.5 μl lipofectamine per ml medium in plate. These tubes were mixed by flicking (vortexing was avoided). Tube A was thoroughly mixed with tube B, incubated at room temperature for 15 min and added dropwise to cells. Cells were incubated with the siRNA/transfection reagent overnight and the medium was completely replaced with fresh growth medium the next day. About 30 h after transfection, the cells were washed with serum/growth-factor-free medium and deprived of serum/growth factors for 18 h (in custom DMEM as described above). These cells were then used in metabolite profiling experiments as described below. See Supplementary Table 1 (siRNA) for the siRNA sequences.
Breast cancer cell line siRNA knockdowns
Cells were seeded in 15 cm plates to reach 70% confluency the next day. Cells were transfected with siRNA as they were seeded, with 20 nM final siRNA mixture and 2.5 μl Lipofectamine per ml final volume in the 15 cm plate. After 24 h, cells were again trypsinized and seeded in 6 cm plates to reach 70% confluency the next day. Cells were seeded in RPMI 1640 medium supplemented with either 5% FBS or 5% dialysed FBS (Life Technologies, 26400-044). Then, 16–20 h later, 3 µM 13C315N1-VB5 was spiked into the medium and tracing experiments were performed as described below.
Snapgene v.5.1 was used for all cloning methodology and processing sequencing data.
A single guide RNA (sgRNA) targeting exon 2 of human PANK2 (Supplementary Table 1 (sdRNA)) was inserted into TLCV2, a pLentiCRISPRv2-based vector modified for doxycycline-inducible expression of Cas9-2A–eGFP, and constitutive expression of the sgRNA and the puromycin resistance marker (Addgene plasmid, 87360, created by the laboratory of A. Karpf38). The guide targeting sequence was identified using E-CRISP39 (http://www.e-crisp.org/). Guide oligos were synthesized by Integrated DNA Technologies and inserted into TLCV2 as previously described for the pLentiCRISPRv2 vector40,41. Plasmids were transformed into NEB Stable competent high-efficiency Escherichia coli (New England Biolabs, C3040H). Clones were sequenced to confirm the insertion. AKT1E17K/+ MCF10A cells were infected with lentivirus containing PANK2 sgRNA-TLCV2 constructs and selected with 1 µg ml−1 puromycin until control cells infected with lentivirus lacking TLCV2 were completely dead. To induce Cas9 expression, cells were maintained in doxycycline for 5 days at 250 ng ml−1, followed by 12 days at 1 µg ml−1. To prepare live cells for GFP-based fluorescence-activated cell sorting (FACS), cells were trypsinized, resuspended in FACS medium (DMEM without phenol red, glucose, glutamine or pyruvate (Thermo Fisher Scientific/Gibco, A1443001), supplemented with 1× penicillin–streptomycin (Thermo Fisher Scientific, 15140163), 5 µg ml−1 plasmocin (InvivoGen, ant-mpt-1), 10 mM HEPES (Thermo Fisher Scientific/Gibco, 15630080), 2% horse serum (Gemini Bio, 100508), 20 mM glucose (Thermo Fisher Scientific, 15023021), 1 mM pyruvate (Sigma-Aldrich, P5280)) and passed through a 40 μm strainer (Thermo Fisher Scientific, 22–363–547) to remove cell clumps. GFP+ cells with the top 25% highest fluorescence intensity (but excluding cells with the highest 5% intensity) were sorted. Cells grown without doxycycline (GFP negative) were used as a negative control for gating. Cells were collected in catch medium (DMEM/F12, 1× penicillin–streptomycin, 5 µg ml−1 plasmocin, 10 mM HEPES, 10% horse serum, 1 mM glutamine and 1 mM pyruvate). Cells were subsequently plated in standard MCF10A growth medium but with 10% horse serum, 10 mM HEPES, 1× penicillin–streptomycin and 5 µg ml−1 plasmocin at limiting dilution (0.5 cells per 100 µl) in 100 µl into 96-well plates with the goal of plating a single cell per well. Single-cell clones were grown into clonal populations and screened for PANK2 expression using western blotting. See Supplementary Table 1 (sgRNA) for the sgRNA sequences.
CRISPR–Cas9-mediated knockouts of PANK4 were created using paired sgRNAs in conjunction with a Cas9 D10A nickase mutant that reduces the probability of off-target double-stranded DNA breaks. Transient transfections of cells were used to introduce the vectors expressing sgRNAs and Cas9 to ensure that the final clones did not express these and could be used to eventually create knockout cell lines stably re-expressing PANK4 with unaltered expression constructs. Paired sgRNAs targeting exon 2 of human PANK4 (Supplementary Table 1 (sgRNA)) were separately inserted into pSpCas9n(BB)-2A-GFP (PX461) (Addgene plasmid, 48140, created by the laboratory of F. Zhang41). The two paired guide sequences were identified using E-CRISP39. Guide oligos were synthesized by Integrated DNA Technologies and inserted into pSpCas9n(BB)-2A-GFP as previously described41. The two vectors (containing one gRNA sequence each) were transiently co-transfected into AKT1E17K/+ MCF10A, AKT1+/+ MCF10A or SUM159 cells using Lipofectamine 2000 (Thermo Fisher Scientific/Invitrogen, 11668027). Then, 2 days after transfection, GFP+ cells were sorted, plated, grown into single-cell clonal populations and screened for PANK4 protein expression as described above for PANK2-KO cells. See Supplementary Table 1 (sgRNA) for the sgRNA sequences.
Site-directed mutagenesis of phosphorylation sites
Site-directed mutagenesis of phosphorylated residues on PANK2 and PANK4 was performed using primers designed with NEBasechanger (New England Bioloabs), the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, E0552S) and NEB Stable competent high-efficiency E. coli (New England Biolabs, C3040H).
Flag-tagged PANK4 expression constructs (pcDNA3.1)
N-terminally Flag-tagged PANK4 (Flag-PANK4) constructs in pcDNA3.1 consisted of the flag-tag sequence (DYKDDDDK), followed by a glycine-serine linker (GS), followed by the sequence of full-length human PANK4 lacking its N-terminal methionine. PANK4 variants included the mutants D623A, D659A, T406A and T406E. Empty pcDNA3.1 was used as a control for transfections. These constructs were transiently transfected into HEK293T cells to produce Flag–PANK4 for in vitro phosphatase assays (see below).
Construction of lentiviral protein expression constructs (pLUIN)
Untagged human PANK2 and PANK4 sequences (accession numbers listed below) were inserted into a pLenti-Ubc-IRES-Neo (pLUIN) lentiviral vector (a gift from S. McBrayer and W. Kaelin42) using the In-Fusion HD Cloning System (Takara/Clontech, 639645). pLUIN consists of a ubiquitin C (Ubc) promotor driving expression of the protein of interest and an internal ribosome entry site (IRES) sequence driving expression of a G418/neomycin-resistance cassette. Except for the neomycin-resistance cassette, pLUIN is the same as the pLenti-Ubc-IRES-hygro construct described previously42. See Supplementary Table 1 (InFusion primers and Sequencing primers) for the sequences.
Lentivirus was generated by transient transfection of the plasmid of interest into HEK293T cells. Cells were plated at 9 × 106 cells per 10 cm plate and transfected with 6.3 μg of the protein-expression plasmid of interest, 11.1 µg psPAX2 packaging plasmid (Addgene plasmid, 12260, created by the laboratory of D. Trono), 0.6 μg pCMV-VSV-G envelope protein plasmid (Addgene plasmid 8454, created by the laboratory of B. Weinberg43) and 3 μl of polyethylenamine (Millipore-Sigma, 408727) per µg of DNA transfected. After 48 h, the medium was collected, run through a 0.45 µm filter (Thermo Fisher Scientific, 09-740-106) and stored in aliquots at −80 °C.
Generation of cell lines with stable expression of PANK2 or PANK4
Cells were infected with lentivirus 24 h after plating at 50% density in medium containing 5 μg ml−1 polybrene (Sigma-Aldrich, 107689). Cells were subsequently selected in either 1 μg ml−1 puromycin (TLCV2) or 500 µg ml−1 G418 (Geneticin; pLUIN).
Heavy-isotope-labelled metabolite labelling and treatments of cultured cells
Cells (600,000–700,000) were plated in 6 cm dishes in standard growth medium such that cells were approximately 75% confluent after 24 h. Three replicate plates for metabolomics and two replicate plates for protein lysates were plated, treated and collected in parallel.
13C315N1-vitamin B5 continuous labelling
VB5-free DMEM was custom formulated based on standard DMEM without pyruvate (Thermo Fisher Scientific/Gibco, 11965) but also without glucose, glutamine and VB5 (custom production by US Biological Life Sciences). Custom DMEM without supplements was used for washes, custom DMEM supplemented with 5 mM glucose, 1 mM glutamine and 3 μM unlabelled VB5 was used for serum/growth factor deprivations, and custom DMEM supplemented with 5 mM glucose, 1 mM glutamine and 3 μM 13C315N1-VB5 was used for tracing. The day after plating (~30 h), cells were washed and then incubated in serum/growth-factor-deprivation medium for 18 h. Fresh serum/growth-factor-deprivation medium was placed on cells for an additional 2 h before washing and incubating cells in tracing medium with 13C315N1-VB5 for 3 h (unless the time point is noted otherwise). Medium changes and treatments were staggered 15 min apart between treatment groups to achieve consistent treatment and collection times.
13C315N1-VB5 pulse-chase labelling
The pulse: serum/growth-factor-deprived cells were first incubated for 4 h with custom DMEM supplemented with 5 mM glucose, 1 mM glutamine and 3 μM 13C315N1-VB5 (the pulse). Cells were then were washed and incubated for 1 h in custom DMEM supplemented with 5 mM glucose and 1 mM glutamine but lacking VB5 to deplete intracellular pools of unincorporated 13C315N1-VB5. The chase: finally, cells were washed in custom DMEM and incubated for 3 h in custom DMEM supplemented with 5 mM glucose and 1 mM glutamine, and either with 3 μM 13C315N1-VB5 or without VB5, and either with 100 nM insulin or vehicle.
For heavy-isotope-labelled 13C315N1-cysteine tracing, treatment medium was prepared from DMEM formulated with 25 mM glucose but without glutamine, methionine, cysteine and cystine (Thermo Fisher Scientific/Gibco, 21013024) that was supplemented with 4 mM glutamine and 100 μM 13C315N1-cysteine (Cambridge Isotope Laboratories, CNLM-3871-H). The plating and treatments were the same as for 13C315N1-VB5 tracing described above except for the medium and supplements used.
Growth factor and inhibitor treatments
For signalling experiments, serum/growth-factor-deprived cells (18 h) were treated with insulin or growth factors for the indicated time periods. Final concentrations and stocks were as follows: insulin (Thermo Fisher Scientific/Gibco, A11382II), final concentration, 100 nM; stock concentration, 100 μM (dissolved in 0.1 N HCl then diluted in water to final concentration); IGF-1 (Thermo Fisher Scientific, 291-G1-01M), final concentration, 50 ng ml−1; stock, 100 µg ml−1 (in 0.1% (w/v) BSA/PBS); and EGF (R&D Systems, 236-EG-01M), final concentration, 50 ng ml−1; stock, 100 µg ml−1 (in 0.1% (w/v) BSA/PBS). Stocks were prepared under sterile conditions, aliquoted and stored at −80 °C. Initiation of growth factor stimulation and isotopic tracing as described above were typically concurrent unless otherwise described.
PI3K and AKT kinase inhibitors
The small-molecule kinase inhibitors used included the PI3K catalytic inhibitors GDC-0941 (Cayman Chemical, 11600)44 and GDC-0032 (Selleck Chemicals, S7103)45 and BYL-719 (Active Biochem, A-1214)46, the AKT catalytic inhibitor GDC-0068 (Selleck Chemicals, S2808)47, and the AKT allosteric inhibitor MK-2206 (Cayman Chemical, 1032350-13-2)48. Note that AKT catalytic inhibitors are known to increase AKT phosphorylation on threonine 308 and serine 473 by stabilizing a conformation in which these phosphorylated residues are inaccessible to phosphatases while, at the same time, fully inhibiting AKT kinase activity and phosphorylation of downstream substrates49. Inhibitor stocks were dissolved in DMSO at 10 mM under sterile conditions, aliquoted and stored at −80 °C. Aliquots were freeze–thawed no more than five times. Insulin/growth factor treatments were preceded by 15 min of pretreatment with DMSO (Thermo Fisher Scientific, BP231-100) or a small-molecule inhibitor dissolved in DMSO. The inhibitors or DMSO were also present in the medium during labelling.
The specific ATP citrate lyase (ACLY) inhibitor NDI-091143 was previously described18. Stocks were dissolved in DMSO at 20 mM under sterile conditions, aliquoted and stored at −80 °C. Working concentrations were 15–20 μM.
The pantothenate kinase inhibitor (Millipore-Sigma, 537983) is equivalent to ‘compound 7’ described in ref. 30. PANK inhibitor stocks were dissolved in DMSO at 10 mM under sterile conditions, aliquoted and stored at −80 °C. Aliquots were freeze–thawed no more than five times. For pantothenate kinase inhibitor treatments, after serum/growth factor deprivation (18 h) as described above, cells were changed into custom VB5-free DMEM supplemented with 5 mM glucose, 1 mM glutamine and no VB5 for 2 h before treatment. Cells were pretreated with the indicated amounts of inhibitor for 15 min and then changed into custom DMEM supplemented with 5 mM glucose, 1 mM glutamine and 100 nM 13C315N1-VB5 plus inhibitor for 3 h.
Metabolomics and heavy-isotope-labelled metabolite tracing
Metabolite extractions from cultured cells
The volumes described here are for 6 cm cell culture dishes unless otherwise noted. Each treatment group within an experiment consisted of five replicate dishes grown, treated and collected in parallel with three dishes for metabolite extraction and two dishes for protein lysates.
80% methanol/water extraction
For unlabelled polar metabolomics analyses, cells were lysed on dry ice in 80% HPLC-grade methanol/20% HPLC-grade water with no ammonium acetate and processed for metabolomics as described below.
80% methanol/water with 1 mM ammonium acetate extraction
For analysis of polar metabolites and acetyl-CoA, the addition of ammonium acetate was found to improve the acetyl-CoA signal in 80% methanol extracts. Cells were washed briefly with 4 ml ice-cold 1× PBS to remove extracellular metabolites and cellular debris. Metabolites (including VB5, CoA and acetyl-CoA) were extracted in 1.7 ml of 4 °C 80% (v/v) HPLC-grade methanol (Sigma-Aldrich, 34860)/20% HPLC-grade water (Sigma-Aldrich, 270733) with 1 mM ammonium acetate (Sigma-Aldrich, 431311) added immediately before use. Extracts were scraped into 2 ml tubes and centrifuged at 21,000g for 10 min at 4 °C to sediment protein and other insoluble material. Supernatants were transferred to 50 ml conical tubes for drying.
10% trichloroacetic acid extraction
For analysis of CoA, acetyl-CoA and other short-chain acyl-CoAs (but not polar metabolites), cells were washed briefly with 4 ml 4 °C 1× PBS to remove extracellular metabolites and cellular debris. Metabolites (including CoA and short-chain acyl-CoAs) were extracted in 1.7 ml of freshly prepared in 4 °C 10% (w/v) trichloroacetic acid (Sigma-Aldrich, T6399) in HPLC-grade water. Extracts were scraped into 2 ml tubes and centrifuged at 21,000g for 10 min at 4 °C to sediment protein and other insoluble material. Supernatants were then passed over an Oasis HLB reversed-phase sorbent column (pre-activated with methanol and pre-equilibrated with water) (Waters, WAT094226), washed with HPLC-grade water and eluted with 2 × 750 µl of HPLC-grade methanol with 25 mM ammonium acetate via gravity flow at room temperature (columns were prepared and washes/elutions carried out according to manufacturer’s instructions). Eluates were collected in 50 ml conical tubes for drying. Adapted from previously described methods12,50.
Sample drying and storage
All samples were dried in 50 ml conical tubes under nitrogen gas at room temperature (typically 2–3 h) using an Organomation 24-position N-EVAP nitrogen evaporator, and dried pellets were stored at −80 °C (samples were always dried down prior to storage).
LC–MS/MS metabolomics analysis
On the day of sample analysis, dried metabolites in 50 ml tubes (from 6 cm dishes) were resuspended in 30 μl HPLC-grade water (Sigma-Aldrich), transferred to 1.5 ml tubes, centrifuged at 21,000g for 2 min at 4 °C and the supernatant was transferred to a fresh 1.5 ml tube. For each sample, half was submitted immediately for MS analysis while the remaining sample was snap-frozen in liquid nitrogen and stored at −80 °C as a backup. For each sample, 5 μl was injected and analysed using a hybrid 5500 or 6500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). The HPLC system used hydrophilic interaction chromatography (HILIC) and a 4.6 mm inner diameter × 10 cm Amide XBridge column (Waters) at 400 μl min−1. Gradients were run starting from 85% buffer B (HPLC-grade acetonitrile) to 42% B from 0–5 min; 42% B to 0% B from 5–16 min; 0% B was held from 16–24 min; 0% B to 85% B from 24–25 min; 85% B was held for 7 min to re-equilibrate the column. Buffer A comprised 20 mM ammonium hydroxide/20 mM ammonium acetate (pH 9.0) in 95:5 water:acetonitrile. Metabolites delivered to the mass spectrometer were analysed using selected reaction monitoring (SRM) performed with positive/negative ion polarity switching. Electrospray ionization (ESI) source voltage was +4,950 V in positive ion mode and −4,500 V in negative ion mode. The dwell time was 3 ms per SRM transition and the total cycle time was 1.39 s. Approximately 10–14 data points were acquired per detected metabolite. Peak areas from the total ion current for each metabolite were integrated using MultiQuant v.3.0 (AB/SCIEX). Metabolite total ion counts for a given transition were normalized to protein content of matched lysates for each treatment group, and treatment replicates were scaled around their replicate group means to normalize for run order effects between replicate groups51. For the unlabelled, targeted metabolomics screen, 283 endogenous water-soluble metabolites were targeted, some in both positive and negative ion mode, for a total of 304 SRM transitions13,52. A significant signal is not detected for all metabolites in every experiment. For heavy-isotope-labelled metabolite tracing, SRMs were adapted from previous studies12,50,52 with optimization of collision energies and the most reliable Q3 product ion if needed. Key SRMs were validated in-house using purified metabolites and/or extracts from cells that had or had not been exposed to labelled metabolites as controls. See Supplementary Table 1 (MS-MS transition) for more information.
Analysis of unlabelled polar metabolites altered between PANK4-KO cell lines with stable re-expression of vector, wild-type or PANK4
MCF10A AKT1p.E17K/+, PANK4-KO reconstituted with empty vector (vector), wild-type PANK4 or PANK4D623A cells were grown for 48 h in serum-free custom VB5-free DMEM supplemented with 10% KnockOut Serum Replacement (Thermo Fisher Scientific/Gibco, 10828028), 10 µg ml−1 insulin, 0.5 mg ml−1 hydrocortisone, 20 ng ml−1 EGF, 100 ng ml−1 cholera toxin, 10 mM glucose, 2 mM glutamine and in the absence of VB5. After 24 h, cells were washed for 1 h in this same medium. After 48 h, cells were seeded into 6 cm plates in the same medium but containing 1 µM VB5. Three replicate plates were seeded for metabolite collection, two for protein collection and three plates that contained medium but no cells as blank controls. Metabolites were collected in 80% (v/v) methanol/water as above at both 48 h and 72 h after plating. For statistical analysis, metabolites were filtered out if the maximum intensity for all treatment group averages was either less than 1.5× the intensity of the average of three blank samples, or was less than 10,000. Metabolite intensities were normalized to relative protein concentrations. Metabolites that were significantly altered in a PANK4-phosphatase-activity-dependent manner were identified by correlation with CoA levels between cell lines (PatternHunter, MetaboAnalyst v.4.0, 2–1–2 for vector–PANK4(WT)–PANK4(D623A), false-discovery rate < 0.1). Of these hits, metabolites altered in the same direction at both time points were analysed using one-way ANOVA (false-discovery rate < 0.1, MetaboAnalyst 4.0). Significantly altered metabolites were visualized by heat map as the fold change relative to vector for each respective time point.
Unlabelled lipidomics analysis
Cell culture conditions and collection of samples for lipidomics
In each experiment, four cell lines were compared with five replicate sample plates per cell line in parallel (three plates for collecting lipids and two plates for collecting protein lysates). The cell lines included MCF10A AKT1p.E17K/+ and MCF10A AKT1p.E17KPANK4-KO cells stably reconstituted with empty vector or full-length untagged wild-type PANK4, PANK4D623A, PANK4D659A, PANK4T406A or PANK4T406E. For comparison of vector, wild-type-, D623A- and D659A-expressing cell lines, cells were seeded in 6 cm plates such that they were around 50% confluent the next day, in serum-free custom VB5-free DMEM supplemented with 10% KnockOut Serum Replacement (Thermo Fisher Scientific/Gibco, 10828028), 10 µg ml−1 insulin, 0.5 mg ml−1 hydrocortisone, 20 ng ml−1 EGF, 100 ng ml−1 cholera toxin, 10 mM glucose, 2 mM glutamine and 3 μM VB5. For comparison of PANK4-KO cell lines expressing vector, wild-type PANK4, PANK4T406A or PANK4T406E cells were grown for 48 h in the above-described medium in the absence of VB5. After 24 h, cells were washed for 1 h in this same medium. After 48 h, cells were seeded into 6 cm plates in the same medium but containing 1 µM VB5. The three replicate plates for lipidomics and two replicate plates for protein lysates were seeded, treated and collected in parallel. Three plates with medium but without cells served as blanks. Plates were changed into fresh medium 24 h after seeding and lipids were extracted 48 h after seeding. For the collection of protein lysates, cells were lysed in NP40 lysis buffer as described in the ‘Immunoblotting (western blotting)’ section. The following lipid collection procedure was adapted from previous methods53. For extraction of lipids, plates were placed on wet ice and quickly washed with 4 °C 1× PBS. Then, 1.5 ml of 80% (v/v) HPLC-grade methanol in HPLC grade water at 4 °C was added to the dishes and cells were scraped into prechilled 15 ml glass vials (Cole-Parmer, EW-34534-10) on wet ice. Once all samples were collected, all of the remaining steps were performed at room temperature. For each sample, 4 ml of HPLC-grade MTBE (methyl tert-butyl ether; Sigma Aldrich, 34875) was added to each glass vial with a glass pipette (Cole-Parmer, EW-2555-3-16), and all of the vials were vortexed for 1 min before rocking for 15 min. To induce phase separation, 0.7 ml of HPLC-grade water was added to each vial and the vials were vortexed for 1 min before centrifugation at 1,000g for 10 min. The final ratio for the MTBE:methanol:water mixture was 10:3:2.5 and the final volume was 6.2 ml. Then, 3.5 ml of the upper (organic) layer from each vial was transferred to a fresh glass vial and dried under nitrogen gas. Dried metabolites were stored at −80 °C until the day of analysis. Immediately before LC–MS/MS analysis, dried samples were resuspended in 35 μl of 1:1 HPLC-grade isopropanol:methanol and centrifuged briefly to collect the liquid in the bottom of the glass tube. The samples were then transferred to a 1.7 ml polypropylene microcentrifuge tube, and centrifuged at 21,000g for 2 min. For each sample, half of the supernatant was transferred to a glass autosampler vial for LC–MS/MS analysis, and the other half was stored in a second glass autosampler vial at −80 °C as a backup.
MS analysis for unlabelled lipidomics
For each resuspended sample, 5 μl was injected into an Agilent 1100-Thermo QExactive Plus-Thermo LipidSearch lipidomics platform. A reverse-phase Cadenza 150 mm × 2 mm C18 column with 3 μm particle size (Imtakt) heated to 40 °C at 240 μl min−1 was used with a 1100 quaternary pump HPLC with room temperature autosampler (Agilent). Lipids were eluted over a 22 min gradient from 32% B buffer (90% isopropyl alcohol (IPA)/10% acetonitrile (ACN)/10 mM ammonium formate/0.1 formic acid) to 97% B buffer. A buffer consisted of 59.9% ACN/40% water/10 mM ammonium formate/0.1% formic acid. Lipids were analysed using a high-resolution hybrid QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) in DDA mode (Top 8) using positive/negative ion polarity switching. DDA data were acquired from m/z 225 to 1,450 in MS1 mode and the resolution was set to 70,000 for MS1 and 35,000 for MS2. MS1 and MS2 target values were set to 5 × 105 and 1 × 106, respectively.
Raw lipidomics data analysis
Raw lipidomics data were analysed for both identification and relative quantification using LipidSearch v.4.1.30 software (Thermo Fisher Scientific) using an internal database of more than 20 main lipid classes and more than 80 subclasses53.
Analysis of lipids altered between PANK4-KO cell lines with stable re-expression of vector, wild-type PANK4, PANK4
D623A or PANK4
Three independent replicate experiments were performed with four cell lines compared in each experiment and three replicate plates per cell line for collecting lipids. Lipid species with any cellular sample values lower than the average of the blanks were filtered out. Remaining lipid species were normalized to the relative protein concentration of the respective treatment group. For each independent experiment, lipid species with differential abundance between cell lines were identified by one-way ANOVA analysis (MetaboAnalyst v.4.0, false-discovery rate < 0.1). For each lipid that passed this criterion in two out of three independent experiments, the fold change in that lipid between vector-expressing cells and the other three cell lines was calculated, and the values from each independent experiment were averaged. In the analysis, replicates with ‘N/A’ readings were treated as ‘no value’. Using an absolute fold change cut-off (vector-expressing cells versus PANK4-WT expressing cells) of 25%, relative changes were tested for correlation to CoA levels in these cell lines (Fig. 4c) (PatternHunter, MetaboAnalyst v.4.0; k-means clustering by sample; pattern: 2–1–2–2 for cell lines empty vector–PANK WT–PANK4D623A–PANK4D659A; false-discovery rate < 0.1)54. Significantly correlated hits were visualized by heat map as average log2-transformed fold change relative to vector-expressing cells.
Analysis of lipids altered between PANK4-KO cell lines with stable re-expression of vector, wild-type PANK4, PANK4
T406A or PANK4
Three replicates between the four cell lines were analysed as above. After analysis using one-way ANOVA, significantly altered lipids (false-discovery rate < 0.1, fold change > 25%) were tested for correlation to CoA levels as described above (PatternHunter, MetaboAnalyst 4.0; pattern: 3–2–1–2 for cell lines empty vector–PANK-WT–PANK4T406A–PANK4T406E; false-discovery rate < 0.1). Significantly correlated lipids with even-chain fatty acids were visualized by heat map as the average fold change relative to the row mean of each lipid.
13C tracing from 13C6-glucose into lipids lipidomics analysis
Cell culture conditions, labelling and collection of samples for labelled lipidomics
Cells were grown for 48 h in custom DMEM in the presence of 10% Knockout Serum Replacement, MCF10A medium growth factors, 10 mM glucose, 2 mM glutamine and in the absence of VB5. Three medium-only samples were plated at the same time as blanks, and one set of samples was grown in unlabelled glucose as a control. After 24 h, cells were washed for 1 h in this same medium. After 48 h, cells were seeded into 6 cm plates in the same medium but containing 1 µM VB5. After 48 h in the presence of VB5, cells were washed in 2 ml of custom medium, and the medium was changed into the same medium but containing 10 mM unlabelled glucose or 10 mM 13C6-glucose (Cambridge Isotope Laboratories, CLM-1396). After 16 h of glucose labelling, lipid extracts were collected as described above. At the time of submission, a quality control sample dilution curve was included with pooled samples diluted at 1×, 0.3× and 0.1× in 50% isopropanol:50% methanol.
Mass spectrometry for 13C-labelled lipidomics
The non-polar lipid samples were resuspended in 35 μl of 1:1 HPLC-grade isopropanol:methanol before LC–MS/MS analysis, 5 μl was injected. A Cadenza 150 mm × 2 mm 3 μm C18 column (Imtakt) heated to 37 °C at 200 μl min−1 was used with a 1200 quaternary pump HPLC with room temperature autosampler (Agilent). Lipids were eluted over a 25 min. Gradient from 32% B buffer (90% IPA/10% ACN/10 mM ammonium formate/0.1 formic acid) to 97% B. A buffer consisted of 59.9% ACN/40% water/10 mM ammonium formate/0.1% formic acid. Lipids were analysed using a high-resolution hybrid QExactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific) in DDA mode (top 8) using positive/negative-ion polarity switching. DDA data were acquired from m/z 225–1,450 in MS1 mode and the resolution was set to 70,000 for MS1 and 35,000 for MS2. MS1 and MS2 target values were set to 5 × 105 and 1 × 106, respectively. Lipidomic data were analysed for both identification and relative quantification using Elements v.2.0 (Proteome Software) using the National Institute of Standards and Technology (NIST) v.17 and Human Metabolome Database (HMDB) v.4.0 small-molecule spectral databases. The Elements software was also used to trace the incorporation of 13C isotopologues into identified lipids as described previously55.
Statistical analysis of labelled lipidomics data
Lipid intensities were normalized to the treatment group protein content. Features were filtered out that had minimum values across experiment samples below the average of three blank medium-only samples. Next, features measured within a linear range were selected for by a correlation coefficient of r ≥ 0.9 between the measured lipid intensities and the expected intensities in the quality control sample dilution curve (1×, 0.3×, 0.1×). Overall, lipid isotopologues of M + 3 and above were enriched in labelled samples relative to unlabelled samples, so features that met this criterion were analysed by t-test between labelled vector and PANK4-WT samples. Lipid species with even-chain fatty acids that were significantly different between treatment groups (false discovery rate < 0.1, fold change > 20%) were visualized by heat map as the fold change relative to the average of the vector samples.
Seahorse XF Cell Mito Stress Test
Cells were grown for 48 h in custom DMEM in the presence of 10% Knockout Serum Replacement, MCF10A medium growth factors, 10 mM glucose, 2 mM glutamine and in the absence of VB5. After 24 h, cells were washed for 1 h in this same medium. After 48 h, cells were passaged into the same medium, but containing 1 µM VB5. After 48 h in the presence of VB5, cells were seeded at 7,500 cells per well in an Agilent XF 96-well plate (Agilent, 101085-004). The Seahorse XF Cell Mito Stress Test was performed on a Seahorse XFe96 Analyzer according to the manufacturer’s instructions, and cells were treated with 1 µM oligomycin, 1 µM FCCP and 500 nM rotenone/antimycin. Oxygen consumption values were normalized to cellular protein content as measured by DC assay.
Cells were grown for 48 h in custom DMEM in the presence of 10% Knockout Serum Replacement, MCF10A medium growth factors, 10 mM glucose, 2 mM glutamine and in the absence of VB5. After 24 h, cells were washed for 1 h in this same medium. After 48 h, cells were seeded into 6 cm plates in the same medium but containing 1 µM VB5. Two sets of plates were seeded in parallel, one to be lysed in hypotonic lysis buffer for acid extraction, and one to be lysed in NP40 lysis buffer as described above. After 48 h in the presence of VB5, the medium was changed. Then, 16 h later, cells were put on wet ice and the medium was aspirated. Cells were washed in 4 °C 1× PBS and then lysed in hypotonic lysis buffer (0.05% NP40, 10 mM HEPES pH 7.4, 10 mM KCl) with protease inhibitor cocktail and scraped into 1.5 ml tubes. Lysates were rocked at 4 °C for 20 min and subsequently centrifuged at 16,000g for 10 min. Supernatant was saved for protein concentration assay and western blot analysis. Pellets were resuspended in 0.2 N HCl and rocked at 4 °C for 20 min and then centrifuged at 16,000g for 10 min. Supernatant was transferred to a fresh tube and neutralized with 1 M Tris-HCl pH 8.0. Acid extracted lysates were normalized to protein levels from non-acid extracted protein lysates. Lysates were also normalized based on total histone H3 levels in an initial immunoblot.
Radioactive 14C-VB5 labelling of cells
For 14C-VB5 labelling experiments, cells were plated to around 70% density in medium with full serum, with three replicates for radioactive labelling measurements, one plate as a radioactivity blank and two plates for protein lysates. All of the plates were treated in parallel as described for heavy-isotope-labelled metabolite tracing experiments except for the use of 100 nCi ml−114C-VB5 (specific activity, 50–60 mCi mmol−1; American Radiolabeled Chemicals, ARC 0653). Plates for protein lysates were collected after 4 h. After 4 h of incubation with 14C-VB5, radiolabelled cells were washed with custom DMEM and then chased for 1 h with custom DMEM containing 5 mM glucose, 1 mM glutamine and no pantothenate. The purpose of this 1 h incubation was to remove free radiolabelled VB5 that had not been incorporated into CoA. Cells were washed with room temperature 1× PBS, collected in 1 ml Solvable Aqueous Based Solubilizer (0.4 M sodium hydroxide in water, plus three specialized surfactants; PerkinElmer, 6NE9100) and incubated at 60 °C for 1 h. Cell extracts were transferred to glass scintillation vials and mixed with 10 ml Ultima Gold Scintillation Fluid (PerkinElmer, 6013321), before measuring disintegrations per minute of 14C on a scintillation counter (Beckman LS 6500). All medium changes and treatments were staggered 15 min apart between treatment arms of a given experiment to achieve consistent treatment and collection times.
Immunoblotting (western blotting)
Cells were washed briefly with 4 °C 1× PBS (Boston BioProducts, BM-220) and collected on wet ice in 4 °C NP40 lysis buffer (1× stock stored at 4 °C: 40 mM HEPES, pH 7.5 (Thermo Fisher Scientific/Gibco, 15630080); 120 mM NaCl (Sigma-Aldrich, S7653); 1 mM EDTA, pH 8.0 (Sigma-Aldrich, E6758); 1% IGEPAL CA-630 (NP40) (Sigma-Aldrich, I3021); 10 mM sodium pyrophosphate tetrabasic (Sigma-Aldrich, S6422); 10 mM β-glycerophosphate (Sigma-Aldrich, G9422S); 50 mM sodium fluoride (Thermo Fisher Scientific, S25547); 5% (v/v) glycerol (Thermo Fisher Scientific, G33–500)) with 1:100 protease inhibitor cocktail (104 mM AEBSF, 80 μM aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin and 1.5 mM pepstatin A) (Sigma-Aldrich, P8340)) added on the day of use. The plates were scraped into 1.5 ml tubes and centrifuged at 15,000g for 10 min at 4 °C. The supernatants were transferred to new tubes and protein content was measured by DC protein assay (based on Lowry assay; Bio-Rad, 5000116). Lysates were normalized for protein concentration and Laemmli sample buffer was added for a final 1× concentration (1×: 50 mM Tris base, pH 6.8 (Thermo Fisher Scientific, BP152); 2% (w/v) SDS (Thermo Fisher Scientific, NC0755841); 5% (v/v) β-mercaptoethanol (Sigma-Aldrich, M3148); 5% (v/v) glycerol (Thermo Fisher Scientific, G33–500); 0.01% (w/v) bromophenol blue; (6× stock aliquots stored at −20 °C and freeze–thawed no more than three times)). Western blot samples were run by SDS–PAGE on 10% Tris-Glycine gels in Tris-glycine running buffer (25 mM Tris Base; 0.1% (w/v) SDS; 250 mM glycine) or 4–12% Bis-Tris gradient gels (Thermo Fisher Scientific, NP0329BOX) in MOPS running buffer (Thermo Fisher Scientific, NP000102). Proteins were transferred to PVDF membranes (Millipore-Sigma, IPVH00010) at a constant 300 mA for 90 min at 4 °C in Tris-glycine transfer buffer (10× Transfer (Electro) Blotting Buffer (Boston BioProducts, BP-190) diluted to 1× (25 mM Tris-base, 192 mM glycine, pH 8.5) in water with 10% methanol (v/v)). Membranes were briefly washed in 1× TBST buffer (20× Tris-buffered saline with Tween-20 (Boston BioProducts, IBB-870) diluted to 1× (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) in water) and blocked in 5% (w/v) non-fat dry milk (Thermo Fisher Scientific, NC9022655) in TBST for 1 h at room temperature. Membranes were briefly washed in TBST and incubated in primary antibody diluted in 5% (w/v) fatty-acid-free bovine serum albumin (BSA) (Boston BioProducts, P-753) in TBST with 0.01% (w/v) sodium azide (Thermo Fisher Scientific) and rocking at 4 °C overnight. Membranes were washed three times for 10 min in TBST at room temperature and incubated in horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 5% (w/v) milk in TBST for 1 h, with rocking at room temperature. Membranes were washed three times for 10 min in TBST at room temperature and incubated in Clarity Western ECL Substrate (Bio-Rad, 1705061) or Clarity MAX Western ECL Substrate (Bio-Rad, 1705062) for 2–5 min. The chemiluminescence signal was detected using film (Thomas Scientific, F-BX810) or a Bio-Rad ChemiDoc Imaging System (Bio-Rad, 17001401).
See Supplementary Table 1 (immunoblot antibodies) for further information regarding antibodies and Supplementary Fig. 1 for raw immunoblot film scans.
Immunoprecipitation analysis of PANK1/2/4
All steps were carried out on wet ice or at 4 °C. Cells in 10 cm dishes were lysed in 700 μl of NP40 lysis buffer on ice and scraped into 1.5 ml tubes. Lysates were centrifuged at 15,000g for 10 min at 4 °C, the supernatants were transferred to a new tube, centrifuged a second time at 15,000g for 5 min at 4 °C and the cleared supernatants were transferred to a new tube. Protein concentrations were normalized and the samples were brought up to the same volume (at least 600 µl) in NP40 lysis buffer. A total of 100 μl of lysate was saved for immunoblotting. The remaining lysate (at least 500 μl) was incubated for 2 h with 1 μg of immunoprecipitation antibody, rocking at 4 °C. Immunoprecipitation antibodies were bound to either protein A/G agarose beads (Thermo Fisher Scientific, 20421) or protein G Dynabeads (Thermo Fisher Scientific, 10004D). Protein A/G agarose beads were washed twice with bead wash buffer (40 mM HEPES, pH 7.5 and 120 mM NaCl), centrifuged at no more than 2,500g for 1 min and resuspended in a 1:1 slurry in bead wash buffer. Protein G Dynabeads were similarly washed and resuspended by pelleting on a DynaMag-2 Magnet (Thermo Fisher Scientific, 12321-D). Then, 20 µl of resuspended beads were added to each immunoprecipitation sample for an additional 2 h with rocking at 4 °C. Three washes were performed with 500 µl NP40 lysis buffer and beads were centrifuged at 2,500g or pelleted using a magnet, followed by removal of all of the supernatant above the settled beads and resuspension in 1× sample buffer. The samples were heated at 95 °C for 5 min and stored at −80 °C. See Supplementary Table 1 (IP antibodies) for further information.
In vitro AKT kinase assay
For in vitro AKT kinase assays, immunoprecipitations using lysates from 10 cm dishes were performed as described above until the final resuspension step. After three washes in NP40 lysis buffer, two further washes were performed in 500 µl kinase reaction buffer (20 mM HEPES, pH 7.5 (Thermo Fisher Scientific/Gibco, 15630080), 10 mM MgCl2 (Sigma-Aldrich), 0.5 mM EGTA (Sigma-Aldrich)). All of the supernatant was removed and the beads were resuspended in 20 µl of kinase reaction buffer +100 µM ATP (Sigma-Aldrich) and 1 mM DTT (Thermo Fisher Scientific) with or without 250 ng of purified GST–AKT1 (Sigma-Aldrich). The samples were incubated for 30 min at 32 °C with periodic agitation and put on ice to stop the reaction. A portion of each sample supernatant was saved and sample buffer was added to 1× final concentration to each supernatant. The beads were resuspended in 1× Laemmli sample buffer. Samples were heated at 95 °C for 5 min and stored at −80 °C. For immunoblotting, approximately one-quarter of each immunoprecipitation sample was loaded per SDS–PAGE gel.
Synthesis of 4′-phosphopantothenate
4′-Phosphopantothenate was prepared by D.B. in the laboratory of J.L.M. See Supplementary Fig. 2 for a diagram of reaction steps for 4′-phosphopantothenate synthesis.
Unless otherwise noted, all reagents were obtained from commercial suppliers and used without further purification. Yields of all reactions refer to the purified products. Silica chromatography was carried out in the indicated solvent system using prepacked silica gel cartridges for use on the Teledyne ISCO Purification System. 1H and 31P NMR spectra were acquired on the Bruker 500 MHz instrument operating at 500 MHz for 1H, 126 MHz for 13C and 202 MHz for 31P. 31P chemical shifts are reported relative to triphenylphosphine oxide at δ 24.7 as an external standard.
Phospho-d-pantothenate was synthesized in three steps from commercially available sodium d-pantothenate as previously described with minor modifications56.
d-Pantothenate benzyl ester
Sodium d-pantothenate (0.500 g, 2.07 mmol) was suspended in dimethylformamide (7.5 ml) and benzyl bromide (0.246 ml, 2.07 mmol) was added. The reaction mixture stirred for 18 h at 70 °C. Solvent was removed under reduced pressure and the resultant residue was dissolved in ethyl acetate (50 ml) and washed with brine (three times with 10 ml). The organic layer was dried over anhydrous sodium sulfate, filtered and condensed under reduced pressure. The crude material was purified by silica flash chromatography (3:1 to 1:0 ethyl acetate:hexanes) to yield d-pantothenate benzyl ester as a colourless oil (0.510 g, 80% yield).
4-Phospho-d-pantothenate tribenzyl ester
Dibenzyl phosphite (1.09 ml, 4.95 mmol) was dissolved in toluene (6.6 ml) and N-chlorosuccinimide (0.661 g, 4.95 mmol) was added. The reaction mixture was stirred at 22 °C for 16 h and then filtered into a dried flask. In a separate dried flask, d-pantothenate benzyl ester (0.51 g, 1.65 mmol) was azeotropically dried with anhydrous pyridine (2 × 5 ml) before being dissolved in anhydrous pyridine (7.4 ml) and cooled to −40 °C (dry ice/acetonitrile). The dibenzylchlorophosphate solution was added dropwise to the d-pantothenate benzyl ester solution at −40 °C. After 2 h at −40 °C, the reaction mixture was maintained at −20 °C for 16 h, after which it was warmed to 22 °C and quenched with water (3 ml). Volatiles were removed under reduced pressure and the resulting residue was dissolved in ethyl acetate (25 ml) and washed sequentially with 1 M H2SO4 (twice with 5 ml) and saturated NaHCO3 (twice with 5 ml). The organic layer was dried over anhydrous sodium sulfate, filtered, and condensed under reduced pressure. Purification by silica flash chromatography (2:1 to 1:0 ethyl acetate:hexanes) yielded 4-phospho-d-pantothenate tribenzyl ester as a colourless oil (0.617 g, 66% yield). 1H NMR (500 MHz, DMSO) δ 7.86 (t, J = 5.9 Hz, 1H), 7.42–7.29 (m, 14H), 5.70 (d, J = 5.7 Hz, 1H), 5.07 (s, 2H), 5.03 (s, 2H), 5.02 (s, 2H), 3.91 (dd, J = 9.3, 4.5 Hz, 1H), 3.79 (dd, J = 9.4, 4.4 Hz, 1H), 3.68 (d, J = 5.7 Hz, 1H), 3.40 (dq, J = 13.2, 6.7 Hz, 1H), 3.28 (dt, J = 12.9, 6.5 Hz, 1H), 2.54 (p, J = 6.9 Hz, 2H), 0.84 (s, 3H), 0.81 (s, 3H). 13C NMR (126 MHz, DMSO) δ 172.04, 171.27, 136.18, 136.12, 136.05, 128.46, 128.42, 128.31, 128.01, 127.98, 127.77, 73.84, 72.96 (d, J = 6.1 Hz), 68.40 (d, J = 5.5 Hz), 65.57, 38.51 (d, J = 8.1 Hz), 34.28, 33.77, 20.39, 19.68. 31P NMR (202 MHz, DMSO) δ −0.42.
4-Phospho-d-pantothenate tribenzyl ester (0.200 g, 0.35 mmol) was dissolved in methanol (2 ml). The flask was flushed with N2 and then Pd/C (10%, 10 mg) was added. The reaction mixture was sparged with H2 for 5 min then stirred vigorously under H2 (1 atm) for 2 h. The reaction mixture was filtered through a 0.45 µm PTFE filter to remove the catalyst and the filtrate was condensed under reduced pressure. The crude product was purified by reverse-phase flash chromatography (C18, 0 to 50% acetonitrile in water) to yield 4-phospho-d-pantothenic acid as an amorphous solid (0.065 g, 62% yield) after lyophilization. 1H NMR (500 MHz, D2O) δ 4.04 (s, 1H), 3.83 (dd, J = 9.6, 4.0 Hz, 1H), 3.63 (dd, J = 9.7, 4.9 Hz, 1H), 3.52 (td, J = 6.5, 3.2 Hz, 2H), 2.64 (t, J = 6.5 Hz, 2H), 0.98 (s, 3H), 0.91 (s, 3H). 13C NMR (126 MHz, D2O) δ 176.13, 174.83, 74.40, 71.37, 52.31, 38.26 (d, J = 7.8 Hz), 34.73, 33.51, 20.57, 18.54. 31P NMR (202 MHz, D2O) δ 0.24. ESI calculated for C9H17NO8P− [M – H]− 298.1, found 298.1.
Synthesis of 4′-phosphopantetheine
4′-Phosphopantetheine was prepared by T.T.Z. in the laboratory of J.L.M. 4′-Phosphopantetheine was prepared from pantetheine through enzymatic phosphorylation using Staphylococcus aureus pantothenate kinase and ATP using an adaptation of a previously described protocol57. Pantetheine was freshly reduced by sodium borohydride before the enzymatic reaction58. The 4′-phosphopantetheine product was purified by reverse-phase HPLC to yield a solid with NMR and LC–MS spectra that were identical to previous reports36,59.
In vitro PANK4 phosphatase assay
Phosphatase assays were adapted from previous methods33. Note that an isolated DUF89 phosphatase domain of PANK4 (C-terminally His-tagged) was used in ref. 33, whereas in this study we used full-length PANK4 (N-terminally Flag-tagged). HEK293T cells were transiently transfected with pcDNA3.1 constructs containing empty vector or Flag-tagged PANK4 for 48 h and lysed as described in the ‘Immunoprecipitation analysis of PANK1/2/4’ section. Anti-flag M2 agarose beads (Millipore-Sigma, A2220) were added to lysates for 4 h with rocking at 4 °C to immunopurify Flag–PANK4. The beads were washed twice in 4 °C 1% NP40 lysis buffer with protease inhibitors but without phosphatase inhibitors, and twice in 4 °C phosphatase reaction buffer (50 mM Tris-HCL pH 7.5, 100 mM NaCl, 0.5 mM Co2+). Excess buffer was removed from above the settled beads before reaction buffer with substrate was added (see below). Immunoprecipitations were run on SDS–PAGE gels alongside BSA standards and gels were Coomassie stained, imaged and quantified using ImageJ to determine Flag–PANK4 protein concentrations.
Reaction using PNPP substrate
Flag-tag immunopurifications on M2 agarose beads were incubated with 20 μl of 50 mM para-nitrophenyl phosphate (PNPP) (New England Biolabs, P0757L) in reaction buffer at 30 °C for 30 min with regular mixing of beads and solution. The reaction was stopped by mixing 10 μl of reaction supernatant with 90 μl of 1 N NaOH. Absorbance at 405 nm was measured on a spectrophotometer.
Reaction using phosphopantothenate or phosphopantetheine substrate
Flag-tag immunopurifications on M2 agarose beads were incubated with 20 μl of water as a control or 2 mM 4′-phosphopantothenate or 4′-phosphopantetheine in reaction buffer at 30 °C for 5 min. Inorganic phosphate released during the reaction was quantified using a malachite green phosphate detection kit (R&D Systems, DY996). Absorbance at 620 nm was measured on a spectrophotometer.
2D proliferation assay
Cells were cultured for 24 h in serum-free custom VB5-free DMEM supplemented with 10% KnockOut Serum Replacement (Thermo Fisher Scientific/Gibco, 10828028), 10 µg ml−1 insulin, 0.5 mg ml−1 hydrocortisone, 20 ng ml−1 EGF, 100 ng ml−1 cholera toxin, 10 mM glucose, 2 mM glutamine and without VB5, before being seeded in the same medium at 7,500 cells per well in 24-well format. Serum, which contains variable concentrations of VB5, was replaced in the medium with KnockOut Serum Replacement, which does not contain VB5, to allow for precise control of VB5 concentrations. The next day (24 to 30 h later), cells were switched to custom DMEM supplemented with nutrients and growth factors as described above, plus 1 μM VB5. Cells were incubated for 4 days with a complete medium change on day 2. On days 0, 2 and 4, cells were fixed through addition of trichloroacetic acid directly to the medium to a final concentration of 8.33% (w/v). Total protein staining was performed by a sulforhodamine B assay60. Raw absorbance at 510 nm was converted to cell number using a standard curve created using MCF10A cells.
3D soft agar colony-formation assay
A 1.25% agar (Thermo Fisher Scientific, BP1423) stock was diluted to 0.625% in standard MCF10A growth medium (see the formula above in the ‘Cell lines and culture conditions’ section), and 2 ml per well on six-well plate was plated as the bottom agar layer. Cells were quickly seeded at 30,000 per well in six-well format, in 2.5 ml of 0.28% agar diluted in standard MCF10A growth medium. After two weeks, cells were stained with 0.01% crystal violet (Sigma-Aldrich, C6158) overnight and imaged on the Bio-Rad ChemiDoc Imaging System (Bio-Rad, 17001401). Colonies were counted manually using ImageJ.
All animal experiments carried out at Beth Israel Deaconess Medical Center (BIDMC) were approved by and performed in accordance with the guidelines of the BIDMC Institutional Animal Care and Use Committee (IACUC) (animal protocol, 102–2015). Mice were maintained in a barrier facility with ventilated cage racks and an automatic watering system. Animal holding rooms were set to maintain a temperature of 70–72°F with 40–60% humidity, and rooms are maintained on a 12 h–12 h day–night cycle from 07:00 to 19:00. ES272 allograft tumour studies were approved by and performed in accordance with the guidelines of the Weill Cornell Medicine IACUC (animal protocol 2013-0116).
ES272 orthotopic mammary allograft tumour mouse model treated with BYL-719
The orthotopic allograft mammary tumour model experiment was performed by B.D.H. at Weill Cornell Medicine. The mouse mammary cancer cell line ES27229, which is also known as FR-161, was previously derived from a mammary tumour arising in a transgenic mouse conditionally expressing mammary-restricted Pik3cap.H1047R (C57BL/6J background)61. The growth of ES272 allograft tumours has been shown to be sensitive to PI3K inhibitor treatments29. ES272 cells were cultured in DMEM plus 10% FBS before injection into mice. To generate orthotopic allograft mammary tumours, 1 × 106 ES272 cells were suspended in serum-free medium, mixed 1:1 with Matrigel and injected into the fourth mammary gland of 8–10 week-old immunocompetent C57BL/6J mice. Once tumours reached a diameter of 0.65 cm, the mice were treated with 45 mg kg−1 of BYL-719 (Alpelisib) or vehicle (0.5% methylcellulose, 0.5% Tween-80) through oral gavage once per day for 10 days. Then 3 h after the final treatment, the mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Tumours were then quickly excised, snap-frozen in liquid nitrogen and stored at −80 °C.
BYL-719 1 h treatment and muscle collection
This mouse experiment was performed at BIDMC in collaboration with the laboratory of J.G.C. BYL-719 (Alpelisib) (Chem Express, HM-206_014-20120614) was dissolved/suspended in vehicle (0.5% methylcellulose, 0.5% Tween-80) at 5 mg ml−1 using a bath sonicator and stored at 4 °C overnight before use. C57BL/6J male mice (Jackson Laboratories, 000664; aged 11–12 weeks) were fed ad libitum with standard chow and then administered 50 mg kg−1 BYL-719 or vehicle through oral gavage. Then, 1 h after treatment, the mice were anaesthetized with avertin and gastrocnemius muscle was collected, immediately snap-frozen in liquid nitrogen and stored at −80 °C.
PANK4 immunoprecipitation from tumours and muscle tissue
Snap-frozen mouse xenograft tumours and skeletal muscle were stored at −80 °C and then kept on dry ice until immediately before processing. Lysates were kept on wet ice. Samples were processed in prechilled (4 °C) NP40 lysis buffer with its standard complement of phosphatase inhibitors (see above) plus 1:50 HALT protease inhibitor cocktail (Thermo Fischer Scientific, 78429). The samples were homogenized in a gentleMACS Dissociator (Miltenyibiotec, 130-096-235) in gentleMACS M tubes (Miltenyibiotec, 130-096-335) on the protein M tube setting. Lysates were cleared by centrifuging the M tubes at 3,000g for 5 min, followed by subsequent centrifugation in 1.5 or 2 ml tubes at 20,000g for 10 min each until the lysates were cleared of insoluble debris. Lysate aliquots were diluted 1:5 or 1:10 before determining protein concentration by DC assay. All immunoprecipitation steps were performed as above, except that tissue lysates were incubated with antibody and beads in NP40 lysis buffer with 1:50 HALT protease inhibitor cocktail.
SUM159 orthotopic mammary xenograft tumour model
This mouse experiment was performed at BIDMC in collaboration with the laboratory of J.G.C. SUM159 cells were maintained in RPMI medium with 10% FBS. Cells tested negative for mycoplasma before injection. On the day of injection, 80% confluent cells were trypsinized, washed and counted. A total of 5 × 106 cells per mouse were resuspended in 100 μl serum-free RPMI medium and placed onto wet ice. Cell suspensions were mixed at a 1:1 (v/v) ratio with Matrigel (Corning, 356230) and orthotopically injected into the fourth mammary fat pad of 6–8 week-old NCr nude female mice (Taconic Biosciences, NCRNU-F). Tumours were measured with callipers three times per week (length and width) for 50 days and tumour volumes were calculated as (width × width × length)/2. Mice were euthanized when the tumours reached either of two end points: a 750 mm3 volume or ulceration. The remaining mice were euthanized at the end of the study. Ten mice were injected per treatment group (one injection per mouse) and the maximum number tumours that progressed to an end point in any single group was four (in the PANK4 knockout + empty vector group). Thus, the top four largest tumours in each treatment group were used for the analysis.
Protein and gene accession numbers
The following human sequences were used for siRNA and gRNA design, protein sequence alignments, and expression in experiments: PANK1, PANK2, PANK3, PANK4. Phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and coenzyme A synthase (COASY). A list of the accession codes is provided in Supplementary Table 1 (Human sequence references).
Alignments of DUF89 domain catalytic aspartates
For amino acid alignments between PANK4 and other DUF89-domain-containing proteins, the species, proteins and corresponding sequence identifiers are as follows: Homo sapiens PANK4 (UniProt: Q9NVE7), Arabidopsis thaliana At2g17340 (UniProt: Q949P3), Pyrococcus horikoshii PH1575 (UniProt: O59272) and Saccharomyces cerevisiae YMR027W (UniProt: Q04371). The catalytic aspartates in the non-PANK4 proteins were identified previously33. Sequences were aligned using Clustal Omega.
Experiments with two treatment groups were analysed using two-tailed Student’s t-tests (α = 0.05). Experiments with three or more treatment groups were analysed using one-way ANOVA with a post hoc Tukey test (α = 0.05). Experiments with three or more treatment groups and multiple metabolites compared as a group were analysed using two-way ANOVA with a post hoc Sidak test (α = 0.05). Data were processed with Excel v.16.46 (Microsoft), MetaboAnalyst v.4.0 and ImageJ v.1.50i (W. Rasband, NIH). Statistical analyses were performed within PRISM 8 graphing software (GraphPad). Biological replicates are individual plates of cells that were plated, treated, collected and analysed in parallel in a single experiment. Experiment replicates are independent experiments with average values of triplicates from each experiment graphed.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.