Fig. 6

LSH and PKM2 activate p53 transcriptional activity to support cell lipid catabolism. a Endogenous PKM2, p53, or LSH were immunoprecipitated from A549 cell lysates treated with 1 μM doxorubicin or untreated for 24 h, and cells were further treated with 100 ng/ml EGF or untreated for 6 h and separated by 10% SDS-PAGE, followed by Western blotting with anti-PKM2, p53 and LSH antibodies. Representative images from three independent experiments are presented. b H1299 cells were treated with 100 ng/ml EGF for 6 h after exogenous PKM2, p53, or LSH was transiently transfected with the corresponding constructs and immunoprecipitated using the indicated antibodies. Representative images from three independent experiments are presented. c HEK293T cells were transiently transfected with p53-Luc promoter plasmid along with vector, EGFP-p53, or GST-PKM2 expression plasmid. After 48 h, the cells were harvested, and p53 luciferase activity was measured (n = 3). Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. d H1299 cells were transiently transfected with p53-Luc promoter plasmid along with vector, EGFP-p53, or GST-PKM2 expression plasmid. After 48 h, the cells were harvested, and p53 luciferase activity was measured (n = 3). Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. e HK1 cells stably transfected with vector or FLAG-LSH were treated with or without doxorubicin (DOX, 1 μM) for 24 h. The cells were transiently transfected with GST-PKM2 or vector for 48 h. After that, the cells were harvested and analysed by immunoblotting. The number indicates the ratio of phosphorylated p53 (Ser15) and phosphorylated p53 (Ser20) to the corresponding total p53 protein in the doxorubicin-treated experiments (control set to 1). Representative images from three independent experiments are presented. f A549 cells stably transfected with shCon or LSH shRNA were treated with or without doxorubicin (DOX, 1 μM). The cells were transiently transfected with PKM2 shRNA or control shRNA for 48 h. After that, the cells were harvested and analysed by immunoblotting. The number indicates the ratio of phosphorylated p53 (Ser15) and phosphorylated p53 (Ser20) to the corresponding total p53 protein in the doxorubicin-treated experiments (control set to 1). Representative images from three independent experiments are presented