Fig. 2: The NGF is regulated by ZBTB46 in prostate cancer cells. | Communications Biology

Fig. 2: The NGF is regulated by ZBTB46 in prostate cancer cells.

From: Nerve growth factor interacts with CHRM4 and promotes neuroendocrine differentiation of prostate cancer and castration resistance

Fig. 2

a ZBTB46 and NGF mRNA levels in LNCaP and C4-2 cells stably expressing an empty vector (EV) or a ZBTB46 cDNA expression vector. * vs. the EV. Data from the quantification of mRNA are presented as the mean ± SEM of three independent experiments; n = 3 per group. ** p < 0.01, *** p < 0.001; by a two-way ANOVA. b Schematic of the predicted ZBTB46-binding element (ZBE) or a non-ZBE and the introduced binding site mutant in the regulatory sequence reporter constructs of human NGF. c ChIP assays of PC3 cells following ZBTB46-knockdown (sh46-2) using a specific antibody against ZBTB46 for IP; NC non-target control. Antibodies against H3K4me3 and IgG, respectively, served as positive and negative controls. Precipitated DNA was quantified via a qPCR of ZBE, non-ZBE, and positive ZBE sites. Enrichment is given as a percentage of the total input. * vs. the NC; # vs. the ZBE. d ChIP assays in C4-2 cells following overexpression of ZBTB46. * vs. the EV; # vs. the ZBE. e, f ChIP assays of C4-2 and LNCaP cells following ZBTB46-knockdown and treatment with charcoal stripped serum (CSS)-containing medium (e) or 10 μM MDV3100 (f) for 48 h. * −CSS vs. +CSS; # vs. the NC. Data from the ChIP assays are presented as the mean ± SEM of three independent experiments; n = 3 per group. **p < 0.01, ***p < 0.001; by a two-way ANOVA. g Relative median fluorescence intensity (MFI) of the wild-type (WT) and mutant (M) NGF reporters in PC3 and NCI-H660 cells. * vs. the WT. h, i WT and M NGF reporter activities in response to ZBTB46 overexpression in C4-2 and LNCaP cells (h) or ZBTB46-knockdown in PC3 and NCI-H660 cells (i). * vs. the EV or NC; # vs. the ZBE WT. j, k Relative MFI of the WT and M NGF reporters in C4-2 and LNCaP cells after treatment with 10 μM MDV3100 (j) and CSS-containing medium (k) for 48 h. * vs. DMSO or −CSS; # vs. the ZBE WT. Data from relative MFIs are presented as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; by a two-way ANOVA.

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