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. 2021 Jan 4;4:22. doi: 10.1038/s42003-020-01549-1

Fig. 5. CHRM4 activates AKT and MYCN signaling and promotes neuroendocrine differentiation of prostate cancer.

Fig. 5

a GSEAs of TCGA prostate cancer dataset showed that higher expression levels of both the NGF and ZBTB46 in prostate cancer tissues were positively associated with a NEPC-responsive gene signature25. NES normalized enrichment score, FDR false discovery rate. b, c Relative mRNA levels of the top 10 candidate genes from Supplementary Table 6 in LNCaP cells with an empty vector (EV) or NGF cDNA vector overexpression (b) or in PC3 cells with non-target control (NC) or NGF shRNA vector expressions (c). d Western blotting of CHRM4 in various prostate cancer cell lines. e Western blotting of CHRM4, CHRM1, CHRM3, p-AKT, AKT, and MYCN in LNCaP cells treated with various concentrations of the NGF protein in charcoal-stripped serum (CSS)-containing medium for 1 week. Fetal bovine serum (FBS)-containing medium without the NGF protein served as a control. f Western blotting of CHRM4, p-AKT, AKT, and MYCN in PC3 cells stably transfected with the NC or CHRM4 shRNA vector, and cells were treated with 100 ng/ml NGF protein in CSS-containing medium for 1 week. g CHRM4, p-AKT, AKT, and MYCN protein levels in C4-2 and C4-2-MDVR cells following stable CHRM4-knockdown. h CHRM4, CHRM1, and CHRM3 protein levels in LNCaP and C4-2 cells cultured in CSS-containing medium for 1 and 2 weeks, followed by treatment with 10 nM dihydrotestosterone (DHT) for 1 day. i Protein levels of CHRM4, p-AKT, AKT, and MYCN in LNCaP and C4-2 cells following stable CHRM4-knockdown and cultured in CSS-containing medium for 1 week. j Relative mRNA levels of CHRM4, CHGA, CHGB, SYP, and ENO2 in PC3 cells following stable expression of NC or CHRM4 shRNA vector and treated cells with 100 ng/ml NGF protein in CSS-containing medium for 1 week. * vs. Phosphate buffered saline (PBS); # vs. the NC. Data from the quantification of mRNA are presented as the mean ± SEM of three independent experiments; n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001; by a two-way ANOVA.