Fig. 5.

SF3B3 regulates SREBF1 via mTOR signaling. A Schematic representation of PCR primers used for the identification of SREBF1a and SREBF1c. B Relative SREBF1a and SREBF1c mRNA levels were examined and analyzed by 3% agarose gel electrophoresis of PCR products. LoVo and HT29 cells were treated with siRNAs for 48 h. C The mRNA expression of SREBF1a and SREBF1c was determined by qRT-PCR. D The immunoprecipitated RNAs by anti-SF3B3 and negative IgG were purified and analyzed by PCR using primers specific for SREBF1 and EZH2. E Schematic diagram illustrating the SREBF1a and SREBF1c promoters. Dual luciferase reporter assay was used to determine SREBF1a and SREBF1c promoter activity in HEK293T cells after infection with shSF3B3#1 lentivirus (shNC vs shSF3B3) or transfection with overexpressing plasmids (empty vector, EV; SF3B3 overexpressing plasmid, SF3B3). F Western blot analysis of full-length SREBF1 and mature SREBF1 (mSREBF1) in SF3B3-knockdown CRC cells. LoVo and HT29 cells were treated with siRNAs for 72 h. G Representative western blots of mTOR, p-mTOR, Raptor, Rictor, p-S6K and p-4EBP1 in SF3B3-knockdown CRC cells. LoVo and HT29 cells were treated with siRNAs for 72 h. H Representative western blots of mTOR and p-mTOR in LoVo xenografts (shNC vs shSF3B3#1). I Representative western blots of mTOR, SREBF1, ACLY, and FASN in SF3B3-knockdown CRC cells after mTOR silencing or overexpression. LoVo and HT29 cells were treated with siRNAs for 12 h, followed by transfection with either simTOR or mTOR-overexpressing plasmids for 60 h. J Schematic diagram illustrating the role of mTOR in regulating SREBF1c in SF3B3-knockdown CRC cells. Data are shown as mean ± SD