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. 2016 Dec 25;8(6):9617–9633. doi: 10.18632/oncotarget.14168

Figure 5. R1881-induction of SEMA3C expression is GATA2-dependent.

Figure 5

We examined R1881-induced expression of SEMA3C in the absence of GATA2 to confirm findings from previous microarray studies showing that knockdown of GATA2 decreases SEMA3C expression. When compared to LNCaP treated with scrambled siRNA (siSCX), knockdown of GATA2 (siGATA2) triggered a significant decrease in basal SEMA3C expression and completely attenuated R1881-mediated dose-dependent induction of SEMA3C as shown by qPCR A. These observations were confirmed at the protein level by Western blot analysis B. of both conditioned media (CM) and whole cell extract (WCE) where total actin served as loading control. In chromatin immunoprecipitation assays, SEMA3C ARE amplicon was shown be enriched in GATA2 immunoprecipitates of lysates from LNCaP cells treated with R1881 as shown by end-point C. and qPCR D. indicating an R1881-dependent recruitment of GATA2 to the SEMA3C intron 2 ARE. Input = 1% input, Isotype = isotype-matched control antibody, IP: GATA2 = GATA2 immunoprecipitates. PCR primers for these experiments were the same as those for Figure 3 and map to the SEMA3C intron 2 ARE. ChIP qPCR values represent a fold enrichment over isotype control of the same treatment condition. Chromatin immunoprecipitation assays previously showing R1881-induced recruitment of AR to the SEMA3C ARE were repeated in the presence of siRNA to GATA2 E. Values represent a fold enrichment over isotype control of the same treatment condition. Data represent mean, ± SD; *p < 0.05, ** p < 0.01, *** p < 0.001.