Fig 2: Activity of GAPDH Is Perturbed in SC-β Cells(A) Quantification of cellular PEP in different GSIS conditions from SC-islets.(B) Western blot of lysates from differentiated SC-β cells and cadaveric islets for GAPDH and PGK1. These human islet lysates were from samples used in GAPDH and PGK activity assays. Each band is a separate preparation of human islets or differentiation of SC-β cells.(C) GAPDH enzyme activity in cadaveric islets, undifferentiated human embryonic stem cells (hES), and differentiated SC-β cells.(D) PGK activity in cadaveric islets, undifferentiated hES cells, and differentiated SC-β cells.(E) GAPDH western blot of lysates from DSS-cross-linking in primary islets, hES cells, and differentiated SC-β cells.(F) GSIS on GAPDH and PGK1-overexpressing SC-β cells.(G) Model of SC-β cell inhibition of GSIS caused by decreased GAPDH activity underlying decreased PEP flux.The p values were calculated using two-way ANOVA with Dunnett’s multiple hypothesis correction. For all experiments shown, color denotes a unique biological replicate within an experiment. Replicates within a biological group are the same color. Each data point in (C) and (D) represents a unique biological replicate for human islets (separate donors), SC-β cells (separate differentiations), and hES cells (different passages). Spaces in western blot panels indicate where images from the same experiment were edited together for ease of presentation. Data are represented as mean ± SD.

Fig 3: Purification of S. gordonii rGAPDH and determination of its enzyme activity. (A) SDS-PAGE gel showing purified fractions of rGAPDH protein from E. coli cell lysates. (1) Uninduced whole cell lysate, (2) Induced whole cell lysate, (3) French pressed cell lysate, (4) Unbound fraction, 5 to 9–100, 150, 200, 250, 300, and 350 mM imidazole eluted fractions, respectively. (B) Western blot of rGAPDH protein against anti-GAPDH antibody (anti rabbit S. gordonii GAPDH). Lanes, 1 and 2 – Purified rGAPDH protein at two different concentrations (3 and 1 μg). (C) Measurement of GAPDH activity in the presence and absence of GAs (treated, 200 μm equal to ∼160 μg/mL and 100 μm equal to ∼80 μg/mL).

Fig 4: Lysine acetylation directly affected the enzymatic activities of proteins involved in glycolysis. Enzyme activities of GAPDH (A), LDH (B), and Eno (C) were measured from crude extracts of the wild-type and the ΔackA mutant at ML and S phase and normalized with total protein (see the section “Materials and Methods”). Effect of acetylation by Ac-P on enzymatic activity assayed in the presence or absence of Ac-P on GAPDH (D), LDH (E) or Eno (F) purified proteins. For LDH and GAPDH, one unit is the amount of enzyme able to generate 1 μmole of NADH per minute (nmole/min/μL). For Eno, one milliunit is the amount of enzyme able to generate 1 nmole of H2O2 per minute (nmole/min/μL). Results are expressed at nmole/min/mg of total protein or μg of purified protein. Results reported are the average of at least three independent experiments. Data represent means ± standard deviation. An asterisk indicates a significant difference using a one-way ANOVA where p < 0.05.

Fig 5: Effects of N3-AZA on GAPDH and GSTP1 protein levels, GAPDH localization and their enzymatic activity. (A) Lysates prepared from N3-AZA (or 0.02% DMSO) treated normoxic and hypoxic FaDu cells were processed for western blotting. N3-AZA treatment did not alter GAPDH and GSTP1 protein levels regardless of O2 conditions. Representative immunoblots and quantitation [mean ± S.E.M.] from three independent experiments are displayed. (B) Cells treated with N3-AZA (or 0.02% DMSO) under normoxia and hypoxia were processed for immunocytochemistry to monitor GAPDH localization; no change in cellular localization of GAPDH was observed in response to N3-AZA treatment. The micrographs are representative of at least three independent experiments; scale bar = 20 μm. (C and D) FaDu cells treated with N3-AZA (or 0.02% DMSO) under normoxia and hypoxia were processed for GAPDH activity assay (C) or GST activity assay (D); the enzymatic activities of GAPDH and GST were significantly reduced only in N3-AZA treated hypoxic cells. Data represent mean ± S.E.M. from three independent experiments.