Fig. 2

ACTA1 is downregulated by PAX3-FOXO1 fusion gene. a Luciferase activity of human ACTA1 gene reporter (546ACTA1) is inhibited by PAX3-FOXO1 in RH30 cells. Left: pLuc-MCS or 546ACTA1 reporter cotransfected RH30 cells in 24-well plate with internal control  pRL-TK and increasing amounts of PAX3-FOXO1 expression plasmid (0, 50, 100, 300 ng). Cells were maintained in DMEM-0.3% FBS-1% P/S for 24-36 h after transfection. Firefly luciferase activity was measured and normalized to Renilla luciferase activty. The reporter activity from the empty expression vector was set to one. Right: Representative assay of PAX3-FOXO1 protein levels by western blot in reporter transfections. b Luciferase activity of human ACTA1 gene reporter (546ACTA1) was repressed by PAX3-FOXO1 fusion gene in RH4 and RH41 cell lines. 546ACTA1 reporter was cotransfected with pRL-TK and increasing amounts of PAX3-FOXO1 expression plasmid (0, 50, 100, 300 ng) into RH4 or RH41 cells. Assay of luciferase activity was performed as in (a). c mRNA expression of ACTA1 in PAX3-FOXO1 overexpressed RH30 cells by qRT-PCR. ACTA1 expression was normalized to human GAPDH levels. Bars represent the mean ± SEM. d ACTA1 protein expression in RH4 cells transfected with increasing amount of PAX3-FOXO1 plasmid. Left: ACTA1 expression levels were assayed by western blot analysis. Right: the relative expression of ACTA1 protein shown on the left. e. Immunofluorescence analysis of ACTA1 expression in RH4 cells with overexpressed PAX3-FOXO1 protein. ACTA1 expression (green), detected to be primarily localized in cytoplasm, was obviously decreased in cells with ectopic PAX3-FOXO1 protein expression. f Knockdown of PAX3-FOXO1 by siRNA oligonucleotides (siPF2, siPF3) increased ACTA1 expression through western blotting. Left: Protein levels of ACTA1 and PAX3-FOXO1 expression assayed by western blot analysis. Right: the quantitative analyses of ACTA1 and PAX3-FOXO1 proteins shown on the left