Fig. 5

TSPAN8 promotes SHH-induced SMO phosphorylation. a, b Flag-PTCH1 (a) or Flag-SMO (b) was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. PLVX, lentiviral stable transfection plasmid pLVX-HA-IRES-Puro. c Endogenous SMO was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. P-Ser, phosphorylated serine. d MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were left untreated or treated with 100 ng/ml SHH. Immunofluorescent studies were performed with the indicated antibodies. e MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were transfected with a GLI1 promoter–luciferase reporter plasmid. These cells were left untreated (left) or treated (right) with 100 ng/ml SHH. Luciferase activity was measured and normalized to Renilla luciferase activity (n = 3 per group). Two-tailed unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001. f, g MCF7 cells with or without expressing GFP-TSPAN8 in the presence or absence of ATXN3 siRNA expression were treated with 20 μM Paroxetine hydrochloride (f) or 2 μM GSK180736A (g) together with 100 ng/ml SHH for 6 h. Cellular extracts were immunoprecipitated with anti-SMO antibody, then immunoblotting analyses tested the serine-phosphorylation expression level. h Histograms show the mean numbers and diameters of spheres formed by MCF7 cells expressing the indicated proteins and ATXN3 shRNA. i MCF7 cells expressing the indicated proteins and ATXN3 shRNA were treated with ADR (left) or PTX (right) with the different concentrations for 24 h. The cell viabilities were determined. Cell numbers with no drug was used as control (n = 3 per group). Two-tailed unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001