Additional file 2 of PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner
Creators: name: Hyroššová, Petra
name: Aragó, Marc
name: Moreno-Felici, Juan
name: Xiarong Fu
name: Mendez-Lucas, Andrés
name: García-Rovés, Pablo M.
name: Burgess, Shawn
name: Figueras, Agnès
name: Viñals, Francesc
name: Perales, Jose C.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Developmental Biology
subject: Cancer

description: Additional file 2: Supplementary Figure 1. HeLa model with stable silencing and overexpression of PEPCK-M. Silencing and overexpression were obtained using lentiviral vectors containing inserts of shRNA against PCK2 or inserts of PCK2 cDNA, respectively. (A) Western blot analysis of mitochondrial and cytosolic PEPCK expression levels in HeLa cells with altered PEPCK-M expression levels: overexpressed (L-PCK2), basal (shCtrl and WT) and knocked down PEPCK-M (sh1-PCK2 and sh2-PCK2). As a positive control of PEPCK-C expression, extracts from mouse liver and Fao hepatoma cells were used. (B) Western blot quantification of PEPCK-M protein abundance in HeLa modified lines. PEPCK-M expression was normalized by gamma tubulin. Results are represented as fold change to HeLa WT. One-way Anova with Sidak multiple comparison post-test analysis indicate significance versus WT. (C) PEPCK-M enzymatic activity in HeLa shCtrl, sh1-PCK2, sh2-PCK2 and L-PCK2 cells grown in basal conditions was measured by production of NADH. One-way Anova with Sidak multiple comparison post-test analysis indicate significance versus shCtrl. Supplementary Figure 2. Time course of glucose depletion under glucose exhaustion conditions. HeLa shCtrl cells were washed 3 times with PBS and treated with DMEM medium containing 1 mM glucose. Concentration of glucose in medium was measured every 3 h, up to 24 h. Supplementary Figure 3. 13C enrichment of serine and glycine in HeLa cells. (A) Cells were exposed to 2 mM [U-13C]glutamine for 4 h in the DMEM media containing 10% dFCS and 25 mM glucose. Incorporation of 13C into proline was analyzed using GC-MS. (B) HeLa shCtrl cells were exposed to 2 mM [U-13C]glutamine for 4 h in the DMEM media containing 10% dFCS and 25 mM glucose or media lacking serine and glycine (MEM) containing 10% dFCS and 5 mM glucose. Incorporation of 13C was analyzed using GC-MS. Negative values were set as 0. Supplementary Figure 4. PEPCK-M inhibition with iPEPCK-2 effects on viability are dependent on Ser/Gly. MTT cell viability assay of HeLa shCtrl cells after 72 h of growth in 0 mM glucose MEM media with or without 0.4 mM serine and 0.4 mM glycine (Ser/Gly). Cells were treated with PEPCK-M inhibitor iPEPCK-2 (5 μM). Fold change was calculated to minus Ser/Gly condition. Significance was determined using two-way Anova with Sidak multiple comparison post-test analysis. Supplementary Figure 5. Glucose content and PEPCK-M activity modulates Proline metabolism. (A) Quantitative real time PCR analysis of PYCR1 and PRODH/POX mRNA expression levels in HeLa cells grown in glucose exhaustion (1 mM) conditions for 24 h. Values were normalized to high glucose (25 mM) condition which is represented by dotted line. Statistical significance to shCtrl was determined by using unpaired two-tailed Student's t-test. (B) 13C enrichment of proline in HeLa cells were exposed to 2 mM [U-13C]glutamine for 4 h in the DMEM media containing 10% dFCS and 25 mM glucose. Incorporation of 13C was analyzed using GC-MS. One-way Anova with Sidak multiple comparison post-test analysis did not show significant differences. (C) Proline effects (5 mM) on viability after iPEPCK-2 treatment (4.3 μM) on HCT116 colon carcinoma cells grown in DMEM media lacking glucose for 48 h. Cell viability was measured using an MTT assay Statistical significance was determined by using unpaired two-tailed Student's t-test. Supplementary Figure 6. Loss of PEPCK-M silencing in xenografts from HeLa cell clones. (A) Tumor growth of HeLa cells implanted into mammary fat pad of BALB/C nude mice. One-way Anova with Sidak multiple comparison post-test analysis did not show significant differences. (B) Western blot analysis of PEPCK-M in tumors from A. Gamma tubulin was used as loading control.