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Fig. 1 | Epigenetics & Chromatin

Fig. 1

From: DNA methylation modifier LSH inhibits p53 ubiquitination and transactivates p53 to promote lipid metabolism

Fig. 1

LSH stabilizes p53. a Endogenous p53 and p21 were detected by Western blotting in CNE1 cells in which LSH or the corresponding vector control was overexpressed. Cell lysates were blotted with the indicated antibodies. Representative images from three independent experiments are presented. b Endogenous p53 and p21 were detected by Western blotting in HK1 cells in which LSH or the corresponding vector control was overexpressed. Cell lysates were blotted with the indicated antibodies. p21 is a p53 target gene. Representative images from three independent experiments are presented. c A549 cell lysates stably expressing LSH shRNA#1, shRNA#2, or control shRNA were blotted with the indicated antibodies. Representative images from three independent experiments are presented. d Overexpression of LSH in CNE1 cell lines has no effect on p53 messenger RNA (mRNA) levels. p53 and LSH mRNA levels were detected by real-time PCR. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. Error bars represent the SEM of triplicate experiments. e Overexpression of LSH in HK1 cell lines has no effect on p53 messenger RNA (mRNA) levels. p53 and LSH mRNA levels were detected by real-time PCR. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. Error bars represent the SEM of triplicate experiments. f Downregulation of LSH in A549 cell lines has no effect on p53 messenger RNA (mRNA) levels. p53 and LSH mRNA levels were detected by real-time PCR. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. Error bars represent the SEM of triplicate experiments. g CNE1 and HK1 cells in which LSH or the corresponding vector control was overexpressed were treated with the proteasome-dependent inhibitor MG132 at 50 μM for 6 h. Next, the cell lysates were blotted with the indicated antibodies. Representative images from three independent experiments are presented. h A549 cells that overexpressed LSH shRNA#1, LSH shRNA#2, or control shRNA were treated with or without the proteasome-dependent inhibitor MG132 at 50 μM for 6 h. Next, the cell lysates were blotted with the indicated antibodies. Representative images from three independent experiments are presented. i, j LSH increases p53 stability. CNE1 cells stably overexpressing LSH or the corresponding vector control were treated with cycloheximide (0.1 mg/ml) and harvested at the indicated times. The left panels show immunoblots of p53 and LSH. The right panel shows quantification of p53 protein levels relative to GAPDH. Representative images from three independent experiments are presented. k, l LSH increases p53 stability. HK1 cells in which LSH or the corresponding vector control were stably overexpressed were treated with cycloheximide (0.1 mg/ml) and harvested at the indicated times. The left panels show immunoblots of p53 and LSH. The right panel shows quantification of p53 protein levels relative to GAPDH. Representative images from three independent experiments are presented. m, n shRNA LSH decreases p53 stability. A549 cells stably expressing shCon or LSH shRNA were treated with cycloheximide (0.1 mg/ml) and harvested at the indicated times. The left panels show immunoblots of p53 and LSH. (SE, short exposure; LE, long exposure.) The right panel shows quantification of p53 protein levels relative to β-actin. Representative images from three independent experiments are presented. j, l, and n Error bars represent the SEM of triplicate experiments

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