Figure 2.

Inhibition of TRIB2 killsenzalutamide-resistantprostate cancer cells via induction of apoptosis.A, cells were plated overnight and treated with gene-specific shRNAs for 4 days. Protein levels of TRIB2, p-Akt, and forkhead box O3-alpha (FOXO3a) were detected by Western blot. B and C, morphological alterations and viability of cells were detected 4 days after shRNA treatment (figure B, the scale bar represents 400 μm). D and E, effect of FANA-modified TRIB2 antisense oligos on LNCaP-ENR tumor growth was tested in nude mice xenografts (n = 3) by intratumoral delivery at 2 mg/kg/day every fourth day. Representative images of tumor-bearing mice from different groups were taken at the end of the study (the scale bar represents 10 mm). The tumor volumes were measured by vernier calipers and presented as mean values ± SE. F, LNCaP-ENR cells were treated with Afatinib (AFA) for 24 h and protein levels were detected by Western blot. G, time-dependent decrease in the viability of LNCaP-ENR cells was measured by MTS/PES assay. H, apoptosis was measured by Annexin V binding after treating LNCaP-ENR cells with 12 μM AFA for 24 h. Positions of the molecular weight markers for the ladder are indicated along with the Western blot data. The data are presented as mean values ± SE. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. For C and G, Two-way ANOVA, Tukey’s multiple-comparison test was applied. FANA, 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid; TRIB2, Tribbles 2.