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. Author manuscript; available in PMC: 2020 Sep 23.
Published in final edited form as: Oncogene. 2020 Mar 23;39(20):4014–4027. doi: 10.1038/s41388-020-1272-x

Figure 2. m1 and m3 are upregulated in CRPC cell lines, and activation of m1 and m3 induces castration resistant growth of PCa cells.

Figure 2.

A, mRNA expression levels of m1 and m3 in 4 different PCa cell lines (LNCaP, MDAPCa2b, 22Rv1 and PC3) measured by qPCR. GAPDH was used for normalization. Compared to LNCaP, MDAPCa2b, 22Rv1, and PC3 had significantly higher mRNA expression of m1. ***P < 0.001 (One-way ANOVA). MDAPCa2b and PC3 had significantly higher mRNA expression of m3 compared to LNCaP. ††P < 0.01, †††P < 0.001 (One-way ANOVA). Bars represent average plus standard error of the mean (SEM) (N = 4). B, Differential response to carbachol measured by pERK expression (pT202/ Y204). Cells are incubated with 100 μM carbachol for 5 min, and subjected to Western blotting. MDAPCa2b, 22Rv1, and PC3 showed increase in pERK with carbachol, but no increase was observed for LNCaP. C, Cell viability in response to carbachol. PCa cells were treated with different concentrations of carbachol for 72 hours. LNCaP did not respond to carbachol, but MDAPCa2b, 22Rv1, and PC3 showed an increase in viability with carbachol. Bars represent average ±SEM (N = 4). ***P < 0.001 (One-way ANOVA). D, Cell viability in response to CNO for LNCaP-DREADD-Gq and 22Rv1-DREADD-Gq cells. One μM of CNO induced increase in cell vitality under 10% FBS, 5% CSS, 5% CSS + 10μM ENZ condition for LNCaP-DREADD-Gq cells, and 1μM of CNO induced increase in cell vitality under 5% CSS, 5% CSS + 10 μM ENZ condition for 22Rv1-DREADD-Gq cells. Bars represent average + SEM (N = 4). *P < 0.05, **P < 0.01 (One-way ANOVA). E, m1, m3, and PSA mRNA expression under CSS + ENZ. LNCaP cells are treated with 5% CSS + 10 μM ENZ for indicated time. m1 or m3 expression increased, and PSA decreased in a time-dependent fashion. Bars represent average + SEM (N = 4). ***P <0.001 (One-way ANOVA). F, Cell viability in response to carbachol before and after CSS + ENZ treatment. After 1 week of 5% CSS + 10 μM ENZ, cells are subjected to viability assay. CSS + ENZ treated cells increased viability with carbachol, but no response was seen for cells under basal conditions cells. Bars represent average ± SEM (N = 4). ***P < 0.001 (One-way ANOVA). G, Response to carbachol before and after CSS + ENZ in LNCaP. pERK increase was seen in CSS + ENZ treated LNCaP with 100μM carbachol, but no difference in pERK was observed for LNCaP cells under basal conditions.