Figure 3
STAT3 mediates the CAMP gene induction by Entinostat.
(a) The effect of the STAT3 inhibitor 5,15-diphenyl-21H,23H-porphine (DPP) at different concentrations (1–50 μM for 24 h) on Entinostat at 2.5 μM induction of the CAMP gene expression in the CampLuc reporter cells. (b) Transcriptional levels of the CAMP mRNA in the parental HT-29 cells after stimulation with 2.5 μM Entinostat (Entino), with or without DPP. Significant reduction vs Entinostat alone is indicated: *p < 0.05 (a,b). (c) Western blot analysis of subcellular fractions i.e. cytoplasm and nucleus, using anti-STAT3 upon induction with Entinostat (2.5 μM, 3 h) in HT-29 cells. GAPDH staining was utilized as a control for normalization. (d) The expression of the STAT3-responsive gene BCL2 analysed by qRT-PCR in HT-29 cells stimulated for 24 h with 2.5 μM Entinostat or untreated (ctrl). Induction vs ctrl: *p < 0.05. (e) HEK293 cells were transfected with either a control vector (shSCR) or with shRNA specifically targeting STAT3 (sh3_STAT3 and sh4_STAT3) transcript. Cells were then stimulated with 2.5 μM Entinostat for 24 h or untreated (ctrl), and analysed for the expression of the genes CAMP, STAT3, and DEFB1 (HBD1) by qRT-PCR, untransfected cells and untreated cells served as controls (ctrl). Significantly altered expression is indicated vs each respective ctrl: *p < 0.05; vs untransfected (untrans) cells stimulated with Entino: §p < 0.05.
