LEGENDS TO SUPPLEMENTARY FIGURES

Fig. S1. BAF53a is mainly nuclear and is found in a complex with BRG1.

(A) Western blot analysis for BAF53a in nuclear and cytoplasmic extracts. Upper panels: RD18 NpBI-206 cells without doxycycline (miR-206 not induced, NI) and after doxycycline treatment for 6 days (miR-206 induced, IND); NIH10T1/2 without doxycycline (MyoD not induced, NI) and after doxycycline treatment for 2 days (MyoD induced, IND); satellite cells in proliferation medium (P) and after 2 days in low serum (D). Tubulin was used as a cytoplasmic marker, HMNG14 was used as a nuclear marker. Lower panels: representative images of MHC immunostaining of the indicated cells. (B) Co-immunoprecipitation assay for either BRG1 or BAF53a in RD18 cells; Immunoprecipitation was done with either anti-BRG1 or anti-BAF53a antibodies, followed by Western blot with anti-BRG1 and anti-BAF53a antibodies.

Fig. S2.Quantification of the percentage of HEK293 and NIH 10T1/2 cells transduced by the bidirectional NpBI 206/GFPlentiviral vector.

FACS analysis for GFP-positive(A) HEK293 and (B)NIH 10T1/2 NpBI 206 cells before and after 3 days of doxycycline induction. In gate R2 the GPP-negative cells, in gate R3 the GFP-positive cells.

Fig. S3. MiR-206 induction is not affected by BAF53a overexpression.

(A-B) Quantitative Real Time PCR for miR-206 in RD18 or RH4 NpBI-206 cells overexpressing either GFP or BAF53a, before and after doxycycline treatment for 6 days (miR-206 not induced, NI; miR-206 induced, IND).

Fig. S4. BAF53a silencing promotes myogenic differentiation in ERMS cells and induces apoptosis in ARMS cells.

Quantification by FACS analysis of(A-B) MHC-positive cells in RD18 and RH4 cells after 6 and 8 days of doxycycline treatment (shRNA BAF53a or shRNA CTRL induced, IND); (C) Apoptotic cells (measured by AnnexinV-allophycocyanine staining) in RD18 and RH4 cells after 5 days of doxycycline treatment (shRNA CTRL or shRNA BAF53a induced, IND). (D-E) Quantitative Real Time PCR for miR-206 in the indicated cells without doxycycline (shRNA CTRL or shRNA BAF53a not induced, NI) or after 6 and 8 days of doxycycline treatment (shRNA CTRL or shRNA BAF53a induced, IND). Mean values (SD) are from 3 independent experiments.

Fig. S5. Conditional silencing of BAF53a with an alternative shRNA impairs ERMS cells proliferation and anchorage-independent growth by inducing myogenic differentiation.

(A) MTT analysis of RD18 cells with inducible expression of a control and an anti-BAF53a shRNA. Cells were analyzed for the indicated days in absence of doxycycline (shRNA not induced, NI) or after doxycycline treatment (shRNA induced, IND). The number of cells at day 0 was set at 100%. (B) Upper panels: representative images of soft agar growth assays of the same cells. Lower panels: quantification of the soft agar growth assays. The number of colonies obtained from the control shRNA cells in NI conditions was set at 100%. Mean values (SD) are from 3 independent experiments. (C) Western blot analysis of the indicated proteins inthe RD18 cells described in A.

Fig. S6. Original Western blots before brightness and contrast adjustments. Molecular weight markers are shown in red.

Table S1. (A) List of primers used for vector construction. (B) Sequences of the mutated 3’UTRs. In green the mutated MREs.

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