Fig. 3

Downregulation of ACTA1 by PAX3-FOXO1 gene is associated with the repression of RhoA-MKL1-SRF signaling pathway. a Luciferase assays of RH30 cells transfected with 546ACTA1 reporter, along with pRL-TK, RhoA (Q63L), MKL1 or SRF in the presence or absence of PAX3-FOXO1 expression plasmid. b. qRT-PCR analysis of ACTA1 in RH30 cells transfected with empty vector, MKL1 or MKL1 and PAX3-FOXO1 plasmids together. c Immunofluorescence analysis of ACTA1 expression using anti-ACTA1 specific antibody in RH30 cells transfected with MKL1 or MKL1 plus PAX3-FOXO1 plasmid. DAPI was used to stain nucleus. ACTA1 protein level was tremendously decreased in cells with PAX3-FOXO1 overexpression. d Luciferase assays of RH30 cells cotransfected with 546ACTA1 reporter and pRL-TK, along with MKL1 and/or STARS in presence or absence of PAX3-FOXO1 expression plasmid. e Luciferase assays of RH30 cells cotransfected with 546ACTA1 reporter and pRL-TK, along with MKL1 in the presence or absence of PAX3-FOXO1 expression plasmid. In the DMEM with serum free starvation, cells were treated with 2 µM Cytochalasin D overnight as indicated and luciferase activities were then examined. f Analysis of ACTA1 protein expression modulated by Cytochalasin D. RH4 cells cotransfected with MKL1, or MKL1 and PAX3-FOXO1 expression plasmid and treated with 2 µM Cytochalasin D were collected and lysed for the determination of ACTA1 expression. Left: Protein levels of ACTA1 and PAX3-FOXO1 assayed by western blot. Right: Relative expression of ACTA1 and PAX3-FOXO1 proteins shown on the left. g Luciferase assays of RH30 cells cotransfected with 546ACTA1 reporter and pRL-TK, along with empty vector, MKL1 and/or PAX3-FOXO1 expression plasmid. As indicated, cells were treated with CCG-1423 at 10 uM for 5 h in the DMEM-0.3%FBS before luciferase assay. h Luciferase assays of RH30 cells cotransfected with 546ACTA1 wild type or CArG box-deleted mutant reporters, along with pRL-TK internal control, empty vector, MKL1 and/or PAX3-FOXO1 expression plasmid. Black bar (■): CArG box