Figure 3: TRIB2-mediated resistance to chemotherapeutics is via AKT activation, suppressing FOXO3a and p53.

(a) Matched isogenic TRIB2 cell line FACS analysis following the knockdown of FOXO3a and subsequent exposure to BEZ235, BAY236 (copanlisib) or BAY439 (BAY1082439) (n=6). P values are indicated for each comparison by two-way ANOVA and data represent the mean±s.d. (b) Quantitative real time PCR (qRT-PCR) analysis of FOXO3a-dependent gene expression after FOXO3a knockdown and isogenic cell line treatment for 24 h with PI3K inhibitors (n=6), (*P≤0.05, **P≤0.01 and were analysed by two-way ANOVA). Data represent the mean±s.d. (c) Matched isogenic TRIB2 cell line FACS analysis after 72 h exposure to various chemotherapeutics (n=6). P values are indicated for each comparison and data represent the mean±s.d. (d) I Representative immunoblot analysis showing 50 μg (MDM2), 100 μg (MDM2-Ser166), 50 μg (p53) total protein lysate per lane separated by 6–10% SDS–PAGE. (e) p53-dependent gene expression was evaluated following TRIB2 isogenic cell line treatment with each indicated chemotherapeutic agent for 24 h. P values are shown for comparison by 2-way ANOVA (*P≤0.05, **P ≤0.001, ***P ≤0.0001) and data shown indicates mean±s.d. (f) Representative immunoblot analysis showing TRIB2 protein expression (100 μg total protein loaded per lane) following exposure to each indicated PI3K inhibitor. (g) FACS analysis of 293T cells after transfection of each TRIB2 construct and subsequent exposure to BEZ235, BAY236 (BAY 80-6946) or BAY439 (BAY1082439) (n=6) for 72 h. P values are indicated for each comparison by two-way ANOVA and data represent the mean±s.d. P values are shown for each comparison where no significant difference was noted. Data was analysed by 2-way ANOVA *P≤0.05, **P≤0.001. (h) Representative immunoblot analysis showing 50 μg (GFP), 100 μg (AKT-Ser473), 100 μg (FOXO3a-Ser253), 100 μg (MDM2-Ser166) protein expression 48 h post-transfection of the indicated GFP tagged TRIB2 plasmid constructs.