Overexpression of TRIB2 in enzalutamide-resistant prostate cancer cells and tumors.A, a model depicting the strategy to develop enzalutamide-resistant prostate cancer cells in vitro. B, heatmap showing upregualted and downregulated genes in LNCaP-ENR cells (EN1 and EN2) compared to parental LNCaP cells (LN1 and LN2). C, volcano plot to show fold change in the levels of gene expression. D, Venn diagram showing unique and common expression of genes in parental and resistant cells. E, upregulation of TRIB2 mRNA in LNCaP-ENR and PCa-2B-ENR cells compared to parental cells by RT-PCR. F, Western blot showing increased protein levels of TRIB2 and targets in enzalutamide-resistant cells, compared to parental cells. Positions of the molecular weight markers for the ladder are indicated along with the Western blot data. G and I, representative IHC pictures (showing average expression) of TRIB2 staining intensity in prostate PDX (n = 20) and patient prostate tumor samples with (n = 53) or without (n = 75) enzalutamide treatment (G, the scale bar represents 100 μm; the scale bar represents 50 μm (inset); I, the scale bar represents 200 μm; the scale bar represents 25 μm (inset)). H and J, dot plot showing quantitation of TRIB2 expression level in prostate tumor samples using QuPath digital image analysis software. The statistical significance of the difference between untreated and treated samples (H and J) was calculated by two-tailed unpaired Student’s t test. IHC, immunohistochemistry; TRIB2, Tribbles 2.