1. The NAD+ Salvage Pathway Supports PHGDH-Driven Serine Biosynthesis
J. Patrick Murphy, Michael A. Giacomantonio, Joao A. Paulo, Robert A. Everley, Barry E. Kennedy, Gopal P. Pathak, Derek R. Clements, Youra Kim, Cathleen Dai, Tanveer Sharif, Steven P. Gygi, Shashi Gujar 
Cell Reports 
Volume 24, Issue 9, Pages e5 (August 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 2381-2391.e5DOI: (10.1016/j.celrep.2018.07.086)
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3. Figure 1 Serine Biosynthesis Is Inhibited by NAD+ Depletion
(A) Schematic of 13C-glucose incorporation into serine and alanine (from pyruvate).
(B) 13C-glucose incorporation into serine in cell lines treated with CI inhibitors (10 μM rotenone, 100 μM phenformin, 1 μM piericidin A, and 100 μM MPP+) or 10 μM NAD+ salvage inhibitor FK866.
(C) 13C-glucose incorporation into alanine following CI or NAD+ salvage inhibition, as in (B).
(D) Time course of 13C-glucose incorporation into serine, alanine, pyruvate, and 3-phosphoglycerate over 48 hr in MDA MB 468 and MDA MB 231 cells treated with DMSO (control), 10 μM FK866, or 10 μM FK866 and 100 μM NMN.
Error bars represent the mean ± SEM. ∗p < 0.05, t test. See also Figure S1 and Data S1.
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4. Figure 2 PHGDH-Dependent Cells Are Sensitive to FK866
(A) Western blotting analysis comparing PHGDH expression in PHGDHhigh and PHGDHlow cell lines, following treatment with 10 μM FK866 with or without 100 μM NMN. MCF-7 lysates are used to bridge between blots.
(B) Confirmed decreases in NAD+ levels by mass spectrometry in both PHGDHlow and PHGDHhigh cell lines treated for 48 hr with 10 μM FK866 and rescued with 100 μM NMN.
(C) Cell counts of PHGDHlow and PHGDHhigh breast cancer cell lines treated with two concentrations of FK866 (10 nM and 10 μM) for 96 hr with or without 100 μM NMN.
(D) 13C-glucose incorporation into serine and alanine in SBP-dependent cell lines treated with 10 μM FK866 and rescued with 100 μM NMN.
Error bars represent the mean ± SEM. ∗p < 0.05, t test. See also Figures S2, S3, and S4.
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5. Figure 3 SBP Dependence Requires NAMPT
(A) Analysis of NAMPT and PHGDH expression by western blotting in NAMPT KD MCF-7 and MDA MB 468 cells.
(B) NAD+ levels in NAMPT KD MCF-7 and MDA MB 468 cells grown in the presence or absence of 100 μM NMN (for 48 hr after puromycin selection).
(C) 13C-glucose incorporation into serine and alanine in NAMPT KD MDA MB 468 cells.
(D) Cell counts in NAMPT KD cells after 96 hr. ns, non-specific (non-targeting).
Error bars represent the mean ± SEM. ∗p < 0.05, t test.
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6. Figure 4 NMN Does Not Rescue PHGDH KD
(A) Western blot for NAMPT and PHGDH expression in PHGDH KD MDA MB 468 cells (grown in the presence or absence of 100 μM NMN for 48 hr after puromycin selection).
(B) 13C-glucose incorporation into serine and alanine in PHGDH KD MDA MB 468 cells.
(C) Cell counts in PHGDH KD MDA MB 468 cells (grown in the presence or absence of 100 μM NMN for 96 hr after puromycin selection).
(D) Western blot for PSAT1, NAMPT, and PHGDH expression in PSAT1 KD MDA MB 468 cells (grown in the presence or absence of 100 μM NMN for 48 hr after puromycin selection).
(E) Cell counts in PSAT1 KD MDA MB 468 cells (grown in the presence or absence of 100 μM NMN for 96 hr after puromycin selection).
(F) 13C-glucose incorporation into serine and alanine in PSAT1 KD MDA MB 468 cells.
(G) A putative model of NAD+ salvage-supported serine biosynthesis. ns, non-specific (non-targeting) shRNA.
Error bars represent the mean ± SEM. ∗p < 0.05, t test.
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7. Figure 5
Effect of NAD+ Salvage Inhibitors on the Incorporation of Glutamine Carbons into α-Ketoglutarate
(A) Schematic of 13C-glutamine incorporation into glutamate and α-ketoglutarate.
(B and C) Shown are the fraction of fully labeled 13C-α-ketoglutarate (B) and 13C-glutamate (C) following 48 hr of FK866 treatment with or without 100 μM NMN.
(D) Western blot comparing PHGDH and NAMPT levels in MCF-7, MDA MB 468, and MDA MB 231 cells.
(E) Western blot of MCF-7 and MDA MB 231 cells overexpressing GFP (GFPOE) or PHGDH (PHGDHOE) compared to MDA MB 468 cells.
(F) In PHGDHOE MDA MB 231 cells, 13C-glucose incorporation into serine following 10 μM FK866 treatment in the presence or absence of 100 μM NMN for 48 hr, compared to the GFPOE cells.
(G and H) In MDA MB 231 cells from (E), the fraction of fully labeled 13C-α-ketoglutarate (G) and 13C-glutamate (H) following 48 hr FK866 treatment in the presence or absence of 100 μM NMN is shown.
Error bars represent the mean ± SEM. ∗p < 0.05, t test. See also Figure S5.
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8. Figure 6
Global Metabolite Profiling following NAD+ Salvage Inhibition and PHGDH KD
(A) Hierarchically clustered heatmap of metabolites following FK866 treatment in PHGDHhigh and PHGDHlow cells (with or without 100 μM NMN) or PHGDH KD in MDA MB 468 cells (for both, mean log2 ratios compared to PBS-treated or ns controls).
(B) Relative taurine, UDP-glucose, and UDP-glucuronate levels across cell lines treated with 10 μM FK866, with or without 100 μM NMN, for 48 hr.
(C) Relative taurine, UDP-glucose, and UDP-glucuronate levels in PHGDH KD and ns shRNA control cells, with or without 100 μM NMN.
(D) A putative model of metabolic differences between PHGDHhigh and PHGDHlow cells highlighting the PHGDH-related and -unrelated effects of elevated NAMPT.
Error bars represent the mean ± SEM. ∗p < 0.05, t test. ns, non-specific (non-targeting) shRNA. See also Figure S6.
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9. Figure 7
Correlation of PHGDH and NAMPT Protein Levels within Breast Cancer Samples
(A) 25 most co-expressed and least co-expressed proteins (by Pearson correlation) with NAMPT in the CPTAC dataset of protein expression across breast cancer samples (n = 105).
(B) Correlation among patients for PHGDH and PSAT with NAMPT in the CPTAC dataset.
(C) Relative protein abundance of PHGDH and NAMPT expression between ER-positive and ER-negative breast cancer samples in the CPTAC dataset.
(D) Top: relative abundance of PHGDH and PSAT1 in patients with the highest or lowest NAMPT expression in the dataset. Bottom: their corresponding tumor subtypes.
(E) Correlation among patients for PHGDH and NAMPT in the METABRIC dataset.
(F) Relative transcript abundance (Z score) of PHGDH and NAMPT expression between ER-positive and ER-negative breast cancer samples in the CPTAC dataset.
See also Figure S7.
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