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. 2016 Sep 7;7(43):70404–70419. doi: 10.18632/oncotarget.11879

Figure 7. Stromal/epithelial interactions are involved in modulating the proliferation of NHPrE1 cells.

Figure 7

A. WST-1 cell proliferation assay to assess proliferation of NHPrE1 cells in the presence or absence of prostatic stromal cells (PrSC). NHPrE1/EV and NHPrE1/AR cells were co-cultured with PrSC for 5 days. While androgen treatment (1 nM R1881) inhibited proliferation of NHPrE1/AR cells in the absence of PrSC, co-culture with PrSC stimulated proliferation of NHPrE1/AR cells and partially reversed the proliferation inhibitory effect of androgens seen in vitro. ** p<0.01, t-test. B. Western blots to assess levels of pSTAT3 in NHPrE1/EV or NHPrE1/AR cells cultured with or without PrSC for 2 days. Levels of pSTAT3 (Tyr-705) and total STAT3 in NHPrE1/EV and NHPrE1/AR cells were compared; beta-Actin served as loading control. Co-culture with PrSC increased the levels of pSTAT3 but not MYC in NHPrE1 cells. C and D. quantification of pSTAT3 Western blot. The levels of pSTAT3 were normalized by total STAT3 (C) or by beta-Actin (D). E. blocking IL-6 attenuated the stimulatory effect of PrSC on NHPrE1 cell proliferation. NHPrE1/AR were cultured with or without PrSC cells in the presence or absence of IL-6 neutralizing antibody for 3 days. Anti-IL-6 attenuated the proliferation stimulation effect of PrSC. * p<0.05, t-test. Similar trend was observed in additional independent experiments. F. WST-1 cell proliferation assay. NHPrE1 cells were cultured in the presence or absence of androgens (1 nM R1881) with or without the addition of IL-6 (25 ng/ml). Addition of IL-6 to the cell culture medium did not induce the proliferation of NHPrE1 cells. G. RT-qPCR to assess expression of IL-6 in PrSC cells. Prostate stromal cells (PrSC) were cultured in the presence or absence of NHPrE1/EV (N/EV) or NHPrE1/AR (N/AR) cells. Co-culture with prostate epithelial cells stimulated production of IL-6 mRNA in PrSC cells. ***p<0.001, t-test.