Fig. 3

PEPCK-M influences proline metabolism. a Concentration of proline in HeLa cells grown for 24 h in high-glucose (25 mM) and glucose exhaustion (1 mM) conditions. Amount of proline was normalized to mg of protein. Concentration was analyzed by LC-MS. Two-way ANOVA with Sidak multiple comparison post-test analysis indicate significance versus shCtrl within media treatment (*), significance between sh1-PCK2 and L-PCK2 within media treatments (#), and significance of media treatment effect ($). b Full ¹³C enrichment of proline in HeLa cells exposed to 2 mM [U-¹³C]glutamine for 4 h in the DMEM media containing 10% dFCS and 0 mM glucose. Full incorporation of ¹³C was analyzed using GC-MS (+4 carbon in GC-MS analysis corresponding to m + 5 on LCMS). One-way ANOVA with Sidak multiple comparison post-test analysis indicate significance versus shCtrl (*), and significance between sh1-PCK2 and L-PCK2 (#). c ¹³C enrichment of proline in HeLa cells was exposed to 25 mM [U-¹³C]glucose for 4 h in the DMEM media containing 10% dFCS. Incorporation of ¹³C was determined by the +2 carbon on GC-MS and corresponded to m + 2 on LCMS. One-way ANOVA with Sidak multiple comparison post-test analysis indicate significance versus shCtrl (*), and significance between sh1-PCK2 and L-PCK2 (#). d, e Quantitative real-time PCR analysis of PYCR1 (d) and PRODH/POX (e) mRNA expression levels in HeLa cells grown for 24 h in high-glucose (25 mM) and glucose exhaustion (1 mM) conditions. Values were normalized to shCtrl for each condition. One-way ANOVA with Sidak multiple comparison post-test analysis indicate significance versus shCtrl (*), and significance between sh1-PCK2 and L-PCK2 (#). f Proline metabolism pathway and its implications for ROS formation. g, h Production of mitochondrial superoxide in HeLa cells treated for 3 h in glucose exhaustion (1 mM) (g) and high-glucose (25 mM) (h) conditions. Superoxide was quantified using MitoSOX fluorescent marker, and mean intensity was measured by flow cytometry. Statistical significance compared with shCtrl was determined by unpaired two-tailed Student’s t test. i Secretion of proline in high-glucose DMEM media (not containing proline) by HeLa cells after 24 h. Concentration of proline was normalized to protein and plotted as % of shCtrl secretion. Concentration was analyzed by LC-MS. Statistical significance between sh1-PCK2 and L-PCK2 was determined by using unpaired two-tailed Student’s t test. j Western blot analysis of p53, p21, and SOD2 in HeLa cells grown for 24 h in high-glucose (25 mM) and glucose exhaustion (1 mM) conditions. Gamma tubulin was used as loading control. k Concentration of TCA cycle intermediates in HeLa cells grown 24 h in high-glucose media (25 mM). Concentration of metabolites was normalized to protein, and fold change was calculated to shCtrl cells. Concentration was analyzed by GC-MS. One-way ANOVA with Sidak multiple comparison post-test analysis indicate significance versus shCtrl. l Cell viability was measured in HeLa cells using an MTT assay after 48 h growth in DMEM media lacking glucose with or without supplementation with proline (5 mM). Data are normalized to shCtrl cells grown in the absence of proline. Statistical significance was determined by using unpaired two-tailed Student’s t test