Fig. 4
TMEM147 promoting the expression of DHCR7 depends on transcription factor STAT2. (a) TheTMEM147-interacting proteins were annotated with their Log10 ratio in Huh7 and HepG2 cell lysate. (b) IP assay showed that Flag-TMEM147-antibody could pull down STAT2 (upper panel). Correspondingly, STAT2-antibody also pulled down TMEM1477 (lower panel). (c) Schematic view of the putative binding sites and sequences of STAT2 on DHCR7 promoter region, predicted by the JASPAR and UCSC websites. (d) STAT2 and p-STAT2 (Tyr690) protein levels in TMEM147 overexpression and silenced and control HCC cells. (e) ChIP-qPCR assay was assessed using anti-STAT2 and anti-IgG antibody to identify STAT2 binding sites on the DHCR7 promoter in the indicated cells. (f) Luciferase reporter gene assays showed that binding of STAT2 to the wild-type DHCR7 promoter significantly increased the transcription of DHCR7. (g) DHCR7 protein levels in STAT2-overexpressing, silenced, and control HCC cells. (h) mRNA levels of TMEM147, DHCR7 and STAT2 relative to those of ACTIN were measured using RT-qPCR. Correlations among TMEM147, DHCR7 and STAT2 in HCC tumor are presented as Pearson’s correlation coefficients. (i) Growth curve assay based on CCK8 analysis; interaction effects between si-STAT2 and TMEM147 on HCC cell proliferation. (j) Representative images of Transwell assay; interaction effects between si-STAT2 and TMEM147 on HCC cell invasion and migration. Scale bars, 100 μm