Fig. 3: Suppression of SNAI2 reduces tumorigenicity and growth, and induces muscle differentiation in vitro and in vivo in FN-RMS.

a Growth curve analysis of RD cells 3 days post puromycin selection after lentiviral infection with shScr or shSNAI2 shRNAs (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). b, c Single cell colony formation assay in RD cells containing shScr or shSNAI2 knockdown and quantitation values of colony forming units (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). d, e Soft agar colony formation assay comparing RD shScr to shSNAI2 infected cells and quantification of colony numbers in wells (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). f RD cells xenografted subcutaneously in mice with shScr (left) or shSNAI2 (right) and followed for 76 days (3 representative mice of 6 shScr, 3 shSNAI2.1, and 3 shSNAI2.2 tumors each, 1 × 10⁶ cells). g Tumor volume of mice injected with either shScr or shSNAI2 cells assessed weekly by caliper measurement represented as mm³ (n = 6 shScr, n = 3 shSNAI2.1, and n = 3 shSNAI2.2 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). h Weight measurement of xenograft tumors with either shScr or shSNAI2.1, SNAI2.2 post mortem (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). i Images of shScr and shSNAI2.2 RD tumors taken at 76 days. j–m Hematoxylin and eosin and Immunohistochemistry of MyHC in the same tumors (Representative images of n = 3 biologically independent experiments.) n Growth of vincristine treated (0.5 mg/kg once weekly for 3 weeks) RD tumors expressing shScr or shSNAI2 assessed by Luciferase imaging. o Tumor volume of transplanted RD xenografts with shScr and shSNAI2 + vincristine (VCR) assessed by caliper measurement represented in mm³ followed for 96 days (n = 10 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). p Tumor weight of RD xenografts expressing shScr or shSNAI2 treated with VCR and harvested at 96 days (n = 20 mice, 20 shScr, 10 shSNAI2.1 and 10 shSNAI2.2 tumors from biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). q Images of shScr and shSNAI2.2 tumors extracted from mice post euthanasia. r–u H&E and MyHC immunohistochemistry of tumor sections from RD xenografts expressing shScr or shSNAI2 treated with vincristine (VCR). Representative images of n = 3 biologically independent experiments. Scale Bars in j, l, r, t = 100 μM, Scale bar in b = 100 μM, d = 10 mm.