Fig. 3. ADT-upregulated NGF is associated with NEPC.

a–c IHC staining (a) and analysis (b and c) of cytoplasmic NGF and nuclear ZBTB46 in prostate cancer tissue sections from patients before and after ADT. The 18 samples were collected from Taipei Medical University-Wan Fang Hospital. Scale bars, 100 µm. Statistical analysis was performed using a two-tailed Student’s t-test. *p < 0.05, ****p < 0.0001. d Western blotting of the NGF, CHGA, and ENO2 in C4-2 and LNCaP cells cultured in charcoal stripped serum (CSS)-containing medium for 1 and 2 weeks, followed by treatment with 10 nM dihydrotestosterone (DHT) for 1 day. e Western blotting for CHGA and ENO2 in C4-2 and LNCaP cells treated with the NGF protein in CSS-containing medium at various concentrations for 1 week. Fetal bovine serum (FBS)-containing medium without the NGF protein served as the control. f Western blotting for NGF, CHGA, ENO2, and ZBTB46 in C4-2 and LNCaP cells stably transfected with an empty vector (EV) or NGF expression vector. g Western blotting for NGF, CHGA, ENO2, and ZBTB46 in PC3 and NCI-H660 cells stably expressing a non-target control (NC) or NGF shRNA vector. h Protein levels of the NGF, CHGA, and ENO2 in NGF-knockdown C4-2 or LNCaP cells and cells treated with CSS-containing medium for 1 week. i, j GSEAs of TCGA prostate cancer dataset showing that high NGF expression levels in prostate tissues were positively associated with a neuronal development signature (KEGG) (i) and a NEPC-responsive gene signature²⁵ (j). NES normalized enrichment score, FDR false discovery rate. k Correlation analysis of NGF with ZBTB46 mRNA levels in clinical tissue samples from the Taylor and TCGA prostate cancer datasets. Significance was determined by a two-way ANOVA.