Fig. 6
From: Caspase-1 cleaves PPARγ for potentiating the pro-tumor action of TAMs
tPPARγ enriches in mitochondria and binds to MCAD and then impairs its activity. a A representative silver stained gel showing examples of the cellular proteins that specifically interact with tPPARγΔ64D (band 1 and 2). The corresponding Coomassie staining gel was shown in Supplementary Figure 28. b, c THP-1 cells were transfected with HA-PPARγ or HA-tPPARγΔ64D plasmids. Cell extracts were immunoprecipitated with anti-HA antibodies, then, analyzed with anti-HA antibodies or anti-MCAD antibodies (b), or the same cell extracts were immunoprecipitated with anti-MCAD antibodies, then, analyzed with anti-MCAD antibodies or anti-HA antibodies (c). MCAD expression or PPARγ expression in two transfected THP-1 cells or nontransfected cells was determined by immunoblot. d, e Extracts of the mitochondrial protein of THP-1 cells that cocultured with MCF-7 for 48 h in the absence or presence of YVAD (d), or transfected with mutant PPARγ (D64A) (e) were analyzed by immunoblot, laminA/C and VDAC1 as the loading control. PPARγ and tPPARγ in whole-cell lysates (WCL) in cocultured THP-1 macrophages was also shown. f LacZ overexpressing THP-1 cells were cultured alone or cocultured with MCF-7 in the absence or presence of YVAD for 24 h, MCAD knockout THP-1 macrophages, tPPARγ overexpression THP-1 macrophages (wild type PPARγ was deleted) and MCAD overexpression THP-1 macrophages were cultured alone for 24 h, then MCAD activity was analyzed by HPLC. Remaining octanoyl coenzyme A was negatively correlated with MCAD activity. Statistical results of total three trials were shown (n = 3). g One representative HPLC result was shown, the peak area of octanoyl coenzyme A was shown (mAU*s). h Peritoneal macrophages were isolated from tumor bearing MMTV-PyMT mice, and TAMs were isolated from tumor bearing MMTV-PyMT mice administration of vehicle or caspase-1 inhibitor YVAD, MCAD activity was analyzed by HPLC. TAMs in each sample were pooled from three mice (n = 3). i One representative HPLC result was shown. All the histograms in this figure show means ± s.e.m., (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, one way ANOVA for multiple comparisons)
