Fig. 2. MrgprF overexpression inhibited tumor cell proliferation.

a Establishment of MrgprF overexpression in A375 cells, verified by Real-time RT-PCR (top) and immunoblot (bottom). Green arrow: exogenous MrgprF-Flag; black arrow: Endogenous MrgprF. b Indicated cell growth was examined by daily counting. c, d MrgprF overexpression reduced BrdU positive staining cells by BrdU incorporation assay in A375 cells. c Quantification data for (d). Scale bar = 50 μm. e MrgprF overexpression impeded the colony formation ability of A375 cells. Quantification data was also shown (right). f, g MrgprF forced expression caused G0/G1 phase arrested cell population as examined by FACS analysis. g Quantification data for (f). h Overexpression of MrgprF decreased the expressions of CDK2, CDK4, CDK6, Cyclin D1, but increased p27 expression. Indicated cell extracts were probed with indicated antibodies. i Establishment of MrgprF knockdown cell lines in A375 cells, verified by Real-time RT-PCR (top) and immunoblot (bottom), respectively. j BrdU incorporation assays were performed in A375 cells. k MrgprF knockdown promoted colony formation abilities of A375 cells. Quantification data is also shown. l–o MrgprF overexpression markedly repressed xenograft tumor growth. Representative xenograft tumor images (l), and quantification data for tumor volumes (m, data are presented as mean ± SEM), tumor masses (n) and mice body weights (o) are shown. p, q Representative IHC staining images of Ki67, Cleaved Caspase 3 (CC3) and phosphorylated form of Akt (p-Akt) for indicated xenograft tumors (p), and the quantification results are also indicated (q). Scale bar = 50 μm. Quantified results for all the immunoblots are indicated below, which are normalized to the β-actin signal, compared to reciprocal control. Bars are the mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Colony no. Colony numbers; Ctrl Scramble control shRNA; sh#1 shRNA#1; sh#2 shRNA#2; pCDH-vec empty vector control; MrgprF ove MrgprF overexpression; HPF High power field