Fig. 2: TCF7L1 upregulates IL-8 and CXCR2 through directly binding to the regulatory sequence of IL-8 and CXCR2.

A Cytokine and chemokine array analysis of PC3 cells stably expressing a nontarget control (NC) or TCF7L1 shRNA vector. Conditioned medium was collected from the supernatant and centrifuged. B, C Intensity of conditioned medium (B) and whole-cell lysates (C) in the cytokine and chemokine array analysis from A. D RT-qPCR showing TCF7L1, IL-8, and CXCR2 mRNA levels in PC3 cells stably expressing the NC or TCF7L1 shRNA vector. E Relative mRNA levels of TCF7L1, IL-8, and CXCR2 in LNCaP and C4-2 cells stably expressing an empty vector (EV) or a TCF7L1 expression vector, by an RT-qPCR. Data from the quantification of mRNA are presented as the mean ± SEM; n = 3 per group. *p < 0.05, **p < 0.01; by a two-way ANOVA. * vs. the EV. F Schematic of the predicted TCF7L1 responsive element (RE) of human IL-8 and CXCR2 regulatory sequences. G, H ChIP assays of LNCaP cells expressing the EV or TCF7L1 cDNA vector with antibodies against TCF7L1 and acetyl-H3 by a pair of primers recognizing indicated TCF7L1-RE sites on IL-8 (G) and CXCR2 (H) regulatory sequences. Enrichment is given as a percentage of the total input and then normalized to IgG. * vs. the EV. I, J Relative mean fluorescent intensities (MFIs) of wild-type (W) and mutant (M) IL-8 and CXCR2 green fluorescent protein (GFP)-reporters in LNCaP cells stably transfected with the EV or TCF7L1 expression vector. K, L Relative MFIs of wild-type IL-8 and CXCR2 GFP-reporters in PC3 cells stably transfected with an NC or TCF7L1 shRNA vector. Quantification of mRNA, ChIP data, and MFIs is presented as the mean ± SEM from three independent experiments. Significance was determined by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.