Fig. 5
Mutant p53 inhibits UCP2 and PGC-1α through the inhibition of SESN1/AMPK signaling. a MDA-MB-231 cells were transduced with lentiviruses containing p53-SH1 or p53-SH2 vectors for mutant p53 silencing or their non-targeting negative control (NT). Left panel: western blotting was performed using 50 μg of whole-protein extracts and probed with the indicated antibodies. The p53 expression was shown as control of p53 knockdown efficacy and the GAPDH expression was used as control of equal proteins loading. Right panel: AMPK was immunoprecipitated from protein extracts using anti-rabbit AMPK antibody (IP: AMPK) and western blot analysis was performed using indicated antibodies. Protein extracts from cells silenced for p53 expression with p53-SH1 or p53-SH2 vectors were also immunoprecipitated with rabbit IgG as control. The blot exhibits equivalent AMPK levels in all samples. b H1299 p53-null cells stably expressing R273H mutant p53 (clone H1) and its respective mock control (clone C9) were used to confirm the regulation of SESN1:AMPK by mutant p53. Left panel: western blotting was performed using 50 μg of whole-protein extracts and probed with the indicated antibodies. Right panel: SESN1 was immunoprecipitated from protein extracts using anti-SESN1 antibody (IP: SESN1) and western blot analysis was performed using indicated antibodies. Protein extracts were also immunoprecipitated with IgG as control. c AsPC1-p53 null cells were transfected with the vectors for the ectopic expression of p53-R273H and its mock control and treated with 1 mM AICA-R for 48 h. Gene expression analysis of the UCP2 and PGC-1α was performed by RT-qPCR and normalized to GAPDH mRNA. #p < 0.05 mock vs mock + AICA-R; *p < 0.05 mock vs R273H and R273H vs R273H + AICA-R. d Panc1 mutR273H-p53 cells were transfected for 48 h with the indicated vectors and their relative negative controls. Gene expression analysis of UCP2 and PGC-1α was performed by RT-qPCR and normalized to GAPDH mRNA. *p < 0.05 shp53 vs CTRL; #p < 0.05 shp53 + DN-AMPK vs shp53 or shp53 + WT-AMPK. e The indicated cell lines were transfected with pRSuper-p53, DN-AMPK, WT-AMPK vectors, or negative controls. ROS levels were analyzed using DCF probe by a multimode plate reader. *p < 0.05 shp53 vs CTRL; #p < 0.05 shp53 + DN-AMPK vs shp53 or shp53 + WT-AMPK. The experiments are representative of three biological replicates
