Fig. 3

TSPAN8 promotes cells stemness by activating Sonic Hh. a qRT-PCR analyses of the mRNA levels of the indicated genes in MCF7 cells with or without TSPAN8 overexpression (n = 3). b Immunoblotting analyses of MCF7 cells with or without TSPAN8 overexpression were performed with the antibodies indicated. GAPDH was used as loading sample control. c qRT-PCR analyses of the mRNA levels of the genes indicated in MCF7 cells with or without TSPAN8 depletion (n = 3). Data represent the mean ± SD of three times of independent experiments. ***P < 0.001. d Immunoblotting analyses of MCF7 cells with or without TSPAN8 depletion were performed with the antibodies indicated. GAPDH was used as loading sample control. e MCF7 cells with or without TSPAN8 overexpression were treated with SHH (100 ng/ml) for 6 h. Immunoblotting analyses and immunoprecipitation were performed with the antibodies indicated. P-Ser, phosphorylated serine. f Luciferase activities in MCF7 cells infected with lentivirus expressing TSPAN8 and GLI1 promoter–luciferase reporter construct were determined. GLI1 promoter reporter activity was normalized by comparison with Renilla luciferase activity (n = 3 per group). Two-tailed unpaired Student’s t test was performed. **P < 0.01. g–j MCF7 cells with or without TSPAN8 overexpression were treated with or without 10 nM Vismodegib for 6 h and immunoblotting analyses were performed (g). qRT-PCR analyses of the mRNA levels were conducted. Data represent the mean ± SD of three times of independent experiments. **P < 0.01 (h). Representative images of tumor spheres are shown. Spheres were cultured for a week before counting. Scale bar = 50 µm (i). Flow cytometry analyses of the ratios of CD44⁺/CD24⁻ in these cells were performed (j)