Figure 7.

Notch3 downregulation in RMS cells reduces tumour growth in vivo. (A) RH-30 cells were transfected with a Notch3 short hairpin (sh)RNA- or control (CTR) shRNA-GFP-plasmids and images of GFP fluorescence (grey) were acquired after 3 weeks of puromycin selection (Left). Arrows depict nuclei of newly formed myofibers (200 × magnification). (Right) 48 h post-shRNAs transfection, positive GFP was separated from negative GFP cells by cell sorting and expression of Notch3^IC, and β-actin (loading control) were analyzed by western blotting. Representative of two independent experiments. (B, Left) Mouse-bearing CTR shRNA (as a mixture of control (CTR) shRNA-GFP/wild-type cells) and Notch3 shRNA (as a mixture of Notch3 shRNA-GFP/wild-type cells) tumour xenografts (black arrows; left flank: a and right flank: f, respectively). (Middle) xenografts from nude mice injected with Notch3 shRNA RH30 cells (wild-type/Notch3 shRNA cell ratio ∼60%/40% f, g, h, i and l). Xenografts from CTR shRNA cells (wild-type/CTR shRNA cell ratio ∼50%/50% a, b, c, d and e) were controls. (Right) Histogram reports tumour volumes of each xenograft. (C, Left) Haematoxylin/eosin staining and immunolabeling of Ki67 and GFP of 5 μm serial sections from xenografts of mice injected with CTR shRNA and Notch3 shRNA RH30 cells (from d and i samples) (400 × magnification). GFP right panels are a higher magnification of left panels (600 × magnification). Representative of three xenografts per condition. (D) Histogram depicts the average of the percentage of GFP-positive cells per field in five fields per tumour section. Bars, S.D. ^*P<0.05. (E) The expression of Notch3^IC was analysed by western blotting in lysates of samples from mice injected with CTR shRNA and Notch3 shRNA RH30 cell suspensions (three xenografts were pooled per group)