Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly

Figure 4 Full-length regenerating gene 4 destablized acetyl-CoA carboxylase 1 and ATP-citrate lyase proteins via proteasomal degradation. A: DLD-1 cells and full-length-regenerating gene 4 (FL-REG4) transfectants were treated with cycloheximide (0.4 μg/mL), followed by western blotting to detect acetyl-CoA carboxylase 1 (ACC1) and ATP-citrate lyase (ACLY) expression; B: DLD-1 cells and FL-REG4 transfectants were treated with MG132 (5 μM, 9 h), followed by western blotting to detect ACC1 and ACLY expression; C: DLD-1 cells, FL-REG4 transfectants, and MG132-treated DLD-1 cells and FL-REG4 transfectants were subjected to proteasomal extract and western blotting to detect ACC1 and ACLY expression; D: Ubiquitin transferases (COP1 E3 ubiquitin ligase, synoviolin 1, Cbl proto-oncogene, NEDD4 like E3 ubiquitin protein ligase were detected by western blotting in DLD-1 cells and FL-REG4 transfectants; E: After co- immunoprecipitation, western blotting was performed in DLD-1 cells and FL-REG4 transfectants, with or without MG132 treatment; F: Nile red staining of DLD-1 cells, FL-REG4 transfectants, and MG132-treated DLD-1 cells and FL-REG4 transfectants; G: Tetrazolium salt assay was performed on DLD-1 cells, FL-REG4 transfectants and MG132- treated DLD-1 cells and FL-REG4 transfectants to detect half maximal inhibitory concentration of 5-fluorouracil and cisplatin. ^aP < 0.001; ^dNo significance. REG4: Regenerating gene 4; ACC1: Acetyl-CoA carboxylase 1; ACCY: ATP-citrate lyase; COP1: COP1 E3 ubiquitin ligase; SYVN1: Synoviolin 1; CBL: Cbl proto-oncogene; NEDD4L: NEDD4 like E3 ubiquitin protein ligase; IgG: Immunoglobulin G.