Fig. 6: SALL4 inhibition impairs SHH-dependent tumor cell growth in vitro.

A-C SALL4 levels have been evaluated in protein lysates of SHH-MB tissues from Gfap-cre/Ptc^fl/fl and Math1-cre/Ptc^fl/fl mice and compared to the cerebella of healthy siblings (Ptc^wt/wt). D Primary SHH-MB cells have been infected with lentiviral particles expressing shSALL4 or shCTR. E Cell proliferation has been measured as Nuclight relative staining calculated at indicated time points by using the IncuCyte® Zoom software. F Transcript levels of SHH target genes and G expression of SHH-related proteins are shown, with H relative densitometric analysis. I SALL4-depleted primary SHH-MB cells, and control cells, have been treated with MGCD0103 (0.5 μM, 24 h) or DMSO as control. Cell proliferation has been measured as relative Nuclight staining calculated using IncuCyte® Zoom software at the indicated time points. J SHH-MB-SLCs neurospheres were dissociated and electroporated with siRNAs to a non-relevant mRNA (siCTR) or murine SALL4 (siSALL4). Representative bright field images of tumor neurospheres were acquired with the IncuCyte® zoom software. Scale bar: 400 µm. K The proliferative capability of SHH-MB-SLCs is expressed as neurospheres-size (μm²). L Protein expression levels of SALL4, GLI1, stemness, and tumorigenic markers are shown with M relative densitometric analysis. Representative immunoblotting of n = 3 biological replicas with similar results are shown in B, G, and L. Vinculin- or actin-normalized densitometric analysis in C, H, and M represent the mean of n = 3 independent experiments ± SD. Data in E and I are normalized to cell scans at time 0 and expressed as FC and represent the mean of n = 3 independent experiments ± SD. Data in F 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 versus siCTR or shCTR calculated using two-sided Student’s t-test. Schematic representations in A and D have been created by BioRender.com.