Fig. 2

Trametinib increases proteasome activity to reduce Id1 levels in KRAS-mutant LUAD cells. A Id1 mRNA expression levels assessed by real-time PCR in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cells treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control). β-ACTIN was used as the reference gene. B GO Biological process (BP) enrichment analysis of murine KRAS-mutant LUAD cells (CMT167 and KLA) treated with trametinib (TRAM) (100 nM) for 72 h or vehicle (Control). C Western blot analysis of Id1 protein levels in KRAS-mutant mouse (left panel) and human (right panel) LUAD cell lines treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control), in the presence or not of the proteasome inhibitor MG-132 (5 μM; 6 h). ß-actin and HSP90 were used as controls for mouse and human cell lines, respectively. D E3 ubiquitin ligases SMURF2 and FBXW7 mRNA expression levels assessed by real-time PCR in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cells treated with (TRAM) (100 nM in murine cells and 500 nM in human cells) for 6 h or vehicle (Control). β-ACTIN was used as the reference gene. E Western blot analysis of the E3 ubiquitin ligases SMURF2 and FBXW7 in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cell lines treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 6 h or vehicle (Control). β-actin was used as control. F Western blot analysis of the E3 ubiquitin ligase SMURF2 and Id1 in KRAS-mutant mouse (left panel) and human (right panel) LUAD cell transduced with lentiviral shRNAs targeting SMURF2 or with a scrambled sequence (Control). Cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control). GAPDH and β-actin were used as control. In Western blot analyses, relative optical density is indicated underneath each lane. Data are expressed as mean ± SD. Comparisons between experimental groups were performed by two-sided t-test (parametric) or Mann–Whitney U-test (non-parametric)