Fig. 6: Expression of MITF and GSK3β in different melanoma lines and effects of Rab7a or TPC2 KO or small molecule blockers. | Nature Communications

Fig. 6: Expression of MITF and GSK3β in different melanoma lines and effects of Rab7a or TPC2 KO or small molecule blockers.

From: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis

Fig. 6

a Representative Western blots for MITF and Rab7 protein expression in different melanoma lines and in the breast cancer line MDA-MB-231, normalized to Vinculin. b, c Correlation plot for MITF/Rab7 expression (b) and statistical analysis of experiments as shown in (a) (mean values ± SEM, n = 5–8). Data points represent biological replicates (c). d Representative images of sections from healthy lymphnode (male, abdomen) and melanoma lymphnode metastasis (male, iliacal) samples stained with hMITF antibody (IHC). Scale bars = 5 mm. e IHC evaluation was carried out considering the percentage of stained tumor cells. Statistical significance was assessed by two-tailed unpaired t-test, *p = 0.0125 (mean ± SD). One dot corresponds to one independent human donor (n = 10 for each condition). Genetic knockout of either Rab7a (f, g) or TPC2 (h, i) in SK-MEL-5 cells shows reduction in the protein levels of MITF and β-Catenin but increased expression of GSK3β. Statistical analysis for the expression levels of MITF, GSK3β, and β-Catenin, WT vs. Rab7a KO or TPC2 KO is shown in (g, i), respectively; significance determined by two-tailed unpaired t-test (g) or by one-way ANOVA (i). Shown are mean values ± SEM, n = 8–14 in (g) and 3–9 in (i). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. j, k Representative blots for MITF protein expression in different melanoma lines after treatment with the TPC2 inhibitor SG-094 (7 µM) or DMSO control for 24 h. Statistical significance was determined by one-way ANOVA. Shown are mean values ± SEM, n = 5–7. *p < 0.05, ***p < 0.001, ****p < 0.0001. Proliferation experiments showing effect of SG094 treatment (7 µM) in SK-MEL-5 and other SK-MEL melanoma lines, significance determined by one-way ANOVA (l) or two-tailed unpaired t-test (m). Shown are mean values ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All n numbers represent biological replicates. Source data are provided as Source Data file.

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