Fig. 7. REST Promotes SLC40A1 expression in TMZ-resistant GSCs via inhibiting LRSAM1 mediated SLC40A1 ubiquitination and degradation.
a Venn chart of REST downstream target screening. b Correlation analysis of REST and SLC40A1 based on TCGA and CGGA datasets. c SLC40A1 expression was correlated with NBT and glioma based on GTEX and TCGA datasets. d SLC40A1 expression was correlated with glioma WHO grades based on TCGA and CGGA datasets. e Kaplan–Meier survival curve for all glioma patients with high and low SLC40A1 expression based on TCGA and CGGA datasets. Western blotting and qPCR assays showing SLC40A1 protein (f) and mRNA expression (g) after REST overexpression and knockdown. h Using UbiBrowser 2.0 found the known E3 ubiquitination ligase of SLC40A1. i Co-IP assays showed the interaction of LRSAM1 and SLC40A1 in U87R and GSCm01R. Western blotting and qPCR assays showing SLC40A1 protein (j) and mRNA expression (k) after LRSAM1 overexpression and knockdown. l Ubiquitination assays showed the SLC40A1 ubiquitination levels in U87R and GSCm01R followed by LRSAM1 knockdown or overexpressed. m The LRSAM1 knockdown or overexpressed U87R and GSCm01R were treated with or without MG132 (50 μM) for 6 hours, and SLC40A1 expression was detected by western blotting. The LRSAM1 knockdown or overexpressed U87R and GSCm01R were treated with CHX (50 μg/ml) and the expression of SLC40A1 protein was detected by western blotting (n, o) and the half-life time (t1/2) was quantitative analysis (p). q, r SLC40A1 expression was detected in U87R and GSCm01R by western blotting after REST overexpressing combined with LRSAM1 overexpressing. s SLC40A1 expression was detected in U87R and GSCm01R by western blotting after REST silencing combined with LRSAM1 silencing. t SLC40A1 expression was detected by western blotting after Erianin treatment combined with REST overexpressed U87R and GSCm01R. All data are shown as the mean ± SD (five independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance.