PRMT5 imposed a dual repression on DDIT3 transcription to promote the malignancy of hepatocellular carcinoma

Figure 4 PRMT5 interacted with STAT3 and recruited it to co-suppress DDIT3 transcription. A: Using JASPAR online database to analyze the binding of STAT3 on DDIT3 promoter; B: The activity of DDIT3 promoter was assessed in HERK293T cells respectively transfected with vector, PRMT5 or PRMT5 and STAT3; C and D: Endogenous PRMT5 interacted with exogenous and endogenous STAT3; E: Localization of PRMT5 and STAT3 protein in HepG2 cells was examined by immunofluorescence assay, scale bar = 25 μm; F: Endogenous PRMT5 interacted with exogenous phospho-STAT3; G: Truncations of STAT3 were constructed as shown in graphic; H: The interaction between PRMT5 and DNA-binding domain was examined by immune-coprecipitation in HEK293T cells; I: The binding of STAT3 on DDIT3 promoter was testified in hepatocellular carcinoma (HCC) cells by chromatin immunoprecipitation (ChIP) assay; J: ChIP-Re-ChIP assay was used to verify that the PRMT5-STAT3 interaction occurred at DDIT3 promoter; K and L: The binding of STAT3 on DDIT3 promoter in PRMT5-overexpressing and knockdown HCC cells was examined by ChIP assay. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. IP: Immunoprecipitation; FL: Full length; shPRMT5: Short hairpin RNA of PRMT5; ChIP: Chromatin immunoprecipitation; IgG: Immunoglobulin G; DAPI: 4’,6-diamidino-2-phenylindole; p-STAT3: Phospho-STAT3; NTD: Amino-terminal domain; CCD: Coiled-coil domain; DBD: DNA-binding domain; LD: Linker domain; SH2: SH2 domain; TAD: Transactivation domain; Scr: Scramble.