Supplementary Figure Legends:
Supplementary Figure 1. Cleaved Notch3 and Notch2 intracellular domains (Notch3IC and Notch2IC) are detected in RMS cell lines. (a) Entire radiographs from western blot analysis of Notch3 levels of expression in nuclear (N) and cytoplasmic (C) -enriched fractions, and in whole-cell (W) lysates. Arrows indicate a band under 100 kDa that was decreased after DAPT treatment in all cell fractions corresponding to Notch3IC. (b) Entire radiographs from western blot analysis of Notch2 levels of expression in nuclear (N, Left) and cytoplasmic (C, Right) -enriched fractions of RD and RH30 cells treated with DAPT (5µM) or vehicle (DMSO) for 72h. Arrows indicate the intracellular domain (IC) of Notch2, that decreases after DAPT treatment in nuclear (N) –enriched fractions. Protein lysates were run trough 7% SDS-PAGE gels. Topoisomerase IIβ and β-actin were used as loading controls to discriminate the different cell fractions.
Supplementary Figure 2. Effects of Notch1 and Notch2 down-regulation in RD and in RH30 cell lines. (a) RD and RH30 cells were transfected with either the most effective Notch1 (upper panel) or Notch2 (lower panel) siRNAs (Hs01_00052328 and Hs_00068799, respectively; Sigma, St Louis, MO) or with a non-targeting control siRNA (CTR siRNA). Western blotting was performed 24h and 48h post-transfection. Radiographs show Notch1IC, Notch1IC (Val1744), Notch2IC, Myogenin, and HES1 expression levels. b-actin was the loading control. 50 mg of protein lysate per lane were loaded for Notch1IC (Val1744) expression. (b) Morphology of RD and RH-30 6 days after treatment with Notch1 siRNA and CTR siRNA. (c) Representative immunofluorescence of RD cells stained with MHC antibody 6 days Notch1, Notch3 and CTR siRNAs post-treatment.
Supplementary Figure 3. Notch3 down-regulation impairs cell proliferation of the alveolar RH41 and the embryonal A204 RMS cell lines. (a) RH41 and A204 RMS cell lines were transfected with a Notch3 siRNA or with a scrambled control siRNA (CTR siRNA), (day 0), and on days 2 and 3 post-siRNA transfection were harvested for counting of living cells by the trypan blue exclusion method. Histograms represent mean values ± standard deviations of three independent experiments done in duplicate. (b) After 24 and 48h from Notch3 and CTR siRNA transfection the expression of Notch3 intracellular domain (Notch3IC), as well as that of β-actin (as loading control), was examined by Western blotting. Autoradiograms are representative of at least 3 independent experiments. *aspecific band.
Supplementary Figure 4. Representative FACS analysis for GFP of RH30 and RD cells infected with an adenovirus expressing HES1 (AdGFP-HES1) or a control adenovirus (AdGFP) 24h post transfection with Notch3 or control (CTR) siRNA. GFP detection was performed 48h post-infection. Living cells left untreated (upper) or transfected with CTR siRNA (middle) or with Notch3 siRNA (lower) were identified by Forward and Side Scatters light through a FACS ARIA II flow cytometer. The frequency of GFP-positive cells with high (GFP High) or low (GFP Low) level of GFP expression and of GFP-negative cell populations was calculated in percentage with a FACS DIVA 6.0 software. Representative of 2 independent experiments.
Supplementary Figure 5. Analysis of RH30 cells transfected with a Notch3 or scrambled control (CTR) short-hairpin (sh)RNA-GFP plasmids. Living RH30 cells left untrasfected (upper) or transfected with CTR shRNA (middle) or with Notch3 shRNA (lower) were identified by Forward and Side Scatters light 48h after transfection (left panels) through a FACS ARIA II flow cytometer. The frequency of high expression and low expression GFP-positive and of GFP-negative cell populations was calculated in percentage with a FACS DIVA 6.0 software (middle and right panels). Representative of 2 independent experiments.
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