Fig 2: AXL is the main mediator of the pro-invasive effect of ST3GAL1.a Western blot of ST3GAL1, AXL, and EMT markers in A375 M6 and MeWo cells transduced as indicated. HSP90 was used as loading control. b, c Matrigel invasion assays in A375 M6 and MeWo cells transduced as indicated. Data are represented as mean ± s.d. P value was calculated by ANOVA and Tukey’s test (n = 3 biological independent experiments). d Representative images of A375 M6 as shown in b. e Average log-transformed normalized expression of AXL, GLI1, SOX2, and ST3GAL1 in each sample. f Heat map of averaged expression of SOX2, ST3GAL1, GLI1, and AXL in normal human neonatal epidermal melanocytes (N) and in patient-derived xenografts (M27, M12, M15). g UMAP visualization of 3322 melanoma patient-derived xenograft cells and normal human neonatal epidermal melanocytes, colored by log-transformed normalized expression of SOX2, GLI1, ST3GAL1, and AXL respectively. Numbers in the plots indicate cell clusters. h Western blot of SOX2, GLI1, ST3GAL1, and AXL in primary and metastatic patient-derived melanoma cells. i Proposed mechanism of action of the SOX2/GLI1-ST3GAL1-AXL axis. ST3GAL1 is transcriptionally regulated by both GLI1 and SOX2 and drives melanoma cell invasion and metastasis. ST3GAL1 induces ligand-dependent AXL dimerization and consequent autophosphorylation at residue Tyr702. Blots in a and h are representative of n = 3 biological independent experiments. HSP90 and GAPDH were used as loading controls. Source data are provided as Source Data file.

Fig 3: Mammalian Grk2 -/- cells exhibit strong loss of Hh signalling upstream of Gli processing AWestern blot analysis of GRK2, GLI1, PTCH1 and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of four different guide RNAs in the presence or absence of SAG. a-tubulin was used as a loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R) (n = 3).B, CHh reporter activity assay for wild-type and Grk2 -/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M), respectively, in the presence or absence of SHH (B). Cells were transfected with mSmoA1-GFP-expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n = 3). Unpaired Student's t-test was used for analysis. ***P < 0.001; **P < 0.01; *P < 0.05 and n.s. (not significant).

Fig 4: Modulation of NUMB in hMB-SLCs. (A) qRT-PCR analysis showed mRNA expression of NUMB isoforms, p66, and p72, and of Hedgehog markers (GLI1, CCND1, HIP1) evaluated in hMB-SLCs CD133+ vs. CD133-. Data represent means ± SD from 3 independent experiments. P-values *p < 0.05. (B) Representative western blot images (left) and densitometric analysis (center) of p66 in hMB-SLCs, after overexpression of p66 vs. Ctrl. ACTIN was used as loading control. (Right) Oncospheres-formation capacity of LvNumb-transduced hMB-SLCs vs. LvCtrl. P-values *p < 0.05. (C) Representative images of western blot (left) and densitometric analysis (right) analysis of p66, NANOG, and GLI1, in hMB-SLCs transfected with siRNA against NUMB (siNUMB) or non-targeting siRNA controls (siCTRL). ACTIN was used as loading control. P-values *p < 0.05. (D) Oncospheres-formation capacity of siNUMB hMB-SLCs vs. siCTRL. P-values vs. siCTRL. Data represent means ± SD from three independent experiments. P-values *p < 0.05. (B–C) Data are means ± SD from three independent experiments. Full-length images are presented in Supplementary Figures.

Fig 5: Numb expression in NSCs. (A) Representative Western Blot (WB) (up) and densitometric analysis (bottom) of endogenous Numb isoforms (p66 and p72) in NSCs grown in selective medium for 5 or 30 days, compared to bulk cells (T0). Actin was used as loading control. P-values *p < 0.05. (B) Representative images of immunofluorescence staining of NSCs for Numb (green) and Gli1 (red); nuclei are counterstained with Hoechst (blue). Scale bar: 10 µm for all panels. (C) qRT-PCR analysis showed mRNA expression of Numb p66, evaluated in NSCs infected with lentivirus carrying Nanog-GFP or Zeo-GFP as control and sorted for GFP. Data represent means ± SD from three independent experiments. P-values *p < 0.05. (D) Representative Western Blot (WB) (left) and densitometric analysis (right) of NSCs before and after in vitro differentiation for 48 h (PDGF); p66, Rbfox3/NeuN, Nanog were evaluated. Actin was used as loading control. P-values *p < 0.05. (A–D) Data are means ± SD from three independent experiments. Full-length images are presented in Supplementary Figures.