Fig. 4

PPARy SUMOylation antagonizes injury after H/R by suppressing NF-κB activation. a H9C2 cells were cultured under normoxic or H/R conditions, and cell lysates were then immunoprecipitated with anti-PPARγ antibody, followed by blotting with anti-SUMO1 or anti-PPARγ antibody. Whole-cell lysates were blotted with anti-SUMO1 antibody. b 293 T cells were cotransfected with HA-SUMO1 and Flag-PPARγ wild-type or SUMO site mutants (K77R and K365R) plasmids as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-HA antibody. c PPARγ-knockdown H9C2 cells were constructed by sh-RNA lentivirus. Sh-PPARγ cells were transfected with vector, PPARγ-WT or K77R plasmids as indicated; then, cells were cultured under normoxic or H/R conditions. Cells lysates were blotted with anti-PPARγ or anti-GAPDH antibody. d TUNEL staining of apoptotic H9C2 cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. e Quantification of apoptotic rates based on TUNEL staining (n = 5). f RT-PCR analysis of IL-1β, IL-6 and TNFα in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions (n = 3). g Western blot analysis of phosphorylated IκBα protein in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions. Quantification of the densitometry of the western blot band is shown above (n = 3). h Representative immunofluorescence images of p65 proteins (green) in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. i Quantification of nuclear p65-positive cells based on TUNEL staining (n = 5). Experiments in a, b, c, f and g were performed three times, and experiments in d, e, h and i were performed five times. Data presented are means ± SD, ^**P < 0.01 vs. sham group, ^##P < 0.01 vs. H/R + Vector group, and ^$$P < 0.01 vs. H/R + PPARy-WT group