Fig. 3

Id1 is involved in the acquired resistance of KRAS-mutant LUAD cells to trametinib treatment. A Western blot analysis of Id1 protein in parental and trametinib resistant (TR) KRAS-mutant mouse (left panel) and human (right panel) cells lines. Cells treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control). ß-actin and HSP90 were used as controls for mouse and human cell lines, respectively. B Flow cytometry analysis of the proportion of apoptotic parental and TR KRAS-mutant mouse (upper panel) and human (lower panel) cells. Parental cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control). Id1 was silenced in TR cells using lentiviral transduced shRNAs (sh1-Id1 and sh2-Id1 in mice and sh-Id1 in human) or with a scrambled sequence (Control) TR-cell lines were cultured in cell media with trametinib (500 nM). Apoptosis was assessed by flow cytometry by Annexin V and 7AAD staining, as previously described [19, 24]. C Upper panel: Western blot analysis of Id1 (17 kDa) and Id1-flag (20 kDa) proteins. Control cells were transfected with a GFP cDNA expressing vector (control). Cells were 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 control. Lower panel: Effects of exogenous Id1 (Id1-flag)-transduction in the survival of KRAS-mutant mouse (CMT167, LLC) and KRAS-mutant human (H1792 and H2009) trametinib-treated tumor cells. Trametinib IC50 is indicated in the figure. D Upper panel: Western blot analysis of Id1 protein in TR KRAS-mutant human cells lines. GAPDH was used as control. Id1 was silenced using the sh1-Id1. The expression of Id1 in sh1-Id1-transduced cells was rescued by retroviral transduction of Id1 cDNA refractory to sh-Id1 inhibition (sh1-Id1/Id1-OE) [24]. Cells treated with Trametinib (TRAM) 500 nM for 72 h or vehicle (Control). Lower panel: Cell viability of the upper panel cells when treated with trametinib (TRAM) 500 nM for 72 h or vehicle (Control). E Western blot analysis of c-Myc, total and phosphorylated p42 MAPK, total and phosphorylated p44 MAPK and FOSL1 in parental and in TR KRAS-mutant human cells lines treated as shown in the upper panel of D. GAPDH was 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 one-way ANOVA followed by the Tukey post hoc test