Fig. 3: H₂O₂ destabilizes FASN and represses lipid synthesis through USP22 in p53^+/+ CRC cells.

A RKO E6 and HT29 cells expressing USP22 shRNAs were lysed and then subjected to immunoblotting using FASN and USP22 antibodies. B 293T cells were transfected HA-USP22 or a control vector, and then treated with CHX and MG132 as indicated. Cell lysates were analyzed by immunoblotting. C RKO E6 cells expressing USP22 shRNA were treated with CHX for the indicated time intervals. Cell lysates were analyzed by immunoblotting. D CRC tissues were double-stained with anti-USP22 and anti-FASN antibodies. Representative images are shown. The blue and white arrows indicate USP22/FASN double positive and double negative areas, respectively. Scale bars, 100 µm. In 10 randomly selected microscope fields of the tumor tissues, the percentages of USP22/FASN double-stained cells and USP22-negative but FASN-positive cells were analyzed and compared (mean ± s.e.m., unpaired Student’s t-test). E RKO cells were transfected with HA-USP22 and then treated with CHX for the indicated time intervals in the presence of H₂O₂ or not. Cell lysates were analyzed by immunoblotting. In B, C, and E, band intensities of FASN were quantified and the results are expressed as FASN levels relative to the control cells (mean ± s.d., n = 3 independent experiments, paired Student’s t-test, right panels). ^∗P < 0.01. F 293T cells were transfected with HA-USP22, Myc-Ubi, and Flag-FASN, and then treated with H₂O_2. Cell lysates were immunoprecipitated using an anti-Flag antibody and then analyzed by immunoblotting. G RKO and HCT116 cells expressing USP22 were treated with H₂O₂ for 24 h. Cellular lipid droplets were visualized fluorescently with Bodipy 493/503. Representative images are shown. Scale bar, 20 µm.