Fig. 4

Basal PDK1-NDRG1 signaling is associated with the response to combined trametinib and dexamethasone. a. A panel of multiple myeloma cell lines was analysed by Western blotting for expression and phosphorylation of NDRG1. Vinculin was used as a loading control. Data are representative of 4 independent experiments. Blot images were cropped for clarity of presentation. b. Quantification of total NDRG1 expression normalised to the vinculin loading control. Significance was determined by one-way Anova *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 4). The magnitude of synergy observed with the combination of trametinib and dexamethasone is indicated (‘-‘ none, ‘+’ low, ‘++’ high). c. MM.1S and MM.1R cells were exposed to a titration of the PDK1 inhibitor GSK2334470 for 5 d. Cell proliferation was assessed by CellTiter-Blue assay. Data are representative of 3 independent experiments. d. MM.1S cells were treated with 3 μM GSK2334470 for 48 h and cell lysates analysed for the indicated proteins by Western blotting. Data are representative of 3 independent experiments. Blot images were cropped for clarity of presentation. e. MM.1S cells were treated with either 0.5% BSA (control) or 100 ng/ml of IGF-1 for 10 min. Cell lysates were analysed by Western blotting for the indicated proteins. Blot images were cropped for clarity of presentation. f. MM.1S cells were treated with either 0.5% BSA or 100 ng/ml IGF-1 and then exposed to either 3 μM GSK2334470, 30 nM trametinib, 100 nM dexamethasone or the combination of trametinib and dexamethasone for 5 d. Cell proliferation was determined by CellTiter-Blue assay. Significance was determined by two-way Anova *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3). g. MM.1S cells were treated with either 0.5% BSA or 3 ng/ml IL-6 and then exposed to either 30 nM trametinib, 100 nM dexamethasone or the combination of trametinib and dexamethasone for 5 d. Cell proliferation was determined by CellTiter-Blue assay. Significance was determined by two-way Anova *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3)