Fig. 5: SALL4 knock-down inhibits the proliferation of SHH-MB cell lines.

Med1-MB cells have been transiently transfected with (A–D) two small interfering RNAs (siRNAs) or (E–H) a short hairpin RNAs (shRNA) targeting SALL4 or a non-relevant control sequence (siCTR or shCTR). Cell proliferation in A and E has been measured as cell confluence calculated using the IncuCyte® Zoom software by phase-contrast images. B, F The transcript levels of SHH signature target genes are expressed as FC versus relative controls. C, G Protein levels of SALL4, GLI1, and AcGLI1 and D, H relative densitometric analysis are shown. I Med1-MB cells have been transiently transfected as indicated, and proliferation is expressed as cell confluence calculated using the IncuCyte® Zoom software by phase-contrast images. J SALL4 protein levels in Med1-MB cells transfected with siREN or siCTR. K qRT-PCR of REN silencing in this setting is shown, as well as L SALL4 densitometric analysis. M SALL4 half-life in Med1-MB cells transfected as indicated and then treated with CHX (100 µg/mL) up to 8 h; densitometric analysis is shown in N. O Med1-MB cells have been transiently transfected as indicated, and proliferation is expressed as cell confluence calculated using the IncuCyte® Zoom software by phase-contrast images. Data in A, E, I, and O are normalized to cell scans at time 0 and expressed as FC, and represent the mean of n = 3 independent experiments ± SD. Representative immunoblotting of n = 3 biological replicas with similar results are shown in C, G, J, and M. Actin-normalized densitometric analysis in D, H, L, and N represent the mean of n = 3 independent experiments ± SD. Data in B, F, and K are normalized to endogenous Gapdh and Hprt control expressed as FC respect to the control sample value and represent the mean of n = 3 independent experiments ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 calculated with two-sided Student’s t-test.