Figure 5. Gα₁₂ regulation of SIRT1 via HIF-1α–mediated induction of USP22.

(A) Inhibition of SIRT1 ubiquitination and degradation by Gα₁₂. SIRT1 immunoprecipitates from HepG2 cells infected with Ad-Gα₁₂QL (or Ad-Con) were immunoblotted for ubiquitin (left) and quantified (middle, n = 3). In another experiment, HepG2 cells were treated with 10 μM cycloheximide for indicated times (right, n = 3). (B) Effect of USP22 gene silencing on inhibition of SIRT1 ubiquitination by Gα₁₂. (C) qRT-PCR assays for Usp22 in the liver (left, n = 5/group) or primary hepatocytes (right, n = 4/group). (D) Luciferase reporter assays for USP22 promoter activity in Gα₁₂-overexpressed AML12 cells. The result shown is combined from 3 independent experiments (n = 6–8 replicates/group for each experiment). Box-and-whisker plot shows median (horizontal lines within boxes), 5%–95% percentile (ends of the boxes), and range of minimum to maximum values (whiskers). Each dot represents an outlying value. Mut1 or Mut2, promoter-reporter constructs with deletion of respective HIF-1α response element sites. (E) Increase in SIRT1 level by Gα₁₂ overexpression through HIF-1α/USP22 axis (right, n = 4/group). (F) Effect of HIF-1α or USP22 gene silencing on SIRT1 induction by Gα₁₂. (G) Effect of RhoA/Rock pathway inhibition on HIF-1α induction by Gα₁₂. (H) Effect of Gα₁₂ overexpression in the liver on HIF-1α/USP22/SIRT1 axis. Immunoblottings were done on the liver homogenates obtained from mice as in Figure 4E. (I) Effect of Gα₁₂ overexpression in hepatocytes on HIF-1α/USP22/SIRT1 axis. Immunoblottings were done on mouse primary hepatocytes infected with Ad-Gα₁₂QL (or Ad-Con) and quantified (n = 3/group). Values represent mean ± SEM. Data were analyzed by 2-tailed Student’s t test (A, C, D, and E) or ANOVA followed by Bonferroni’s post hoc test (I). For A, B, and E–I, blots in each panel were run in parallel using the same samples and β-actin was used as a normalization control for densitometric analysis.