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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Cancer Discov. 2021 Jan 11;11(5):1118–1137. doi: 10.1158/2159-8290.CD-20-0751

Figure 2: CBP and p300 protein knockdown impacts AR-V7 expression and AR signaling independent of C-MYC expression in castration resistant prostate cancer models.

Figure 2:

(a-c) 22Rv1 and (d-f) LNCaP95 were transfected with a total of 100nM siRNA with 50nM of either CBP, p300, p300 plus CBP, or C-MYC siRNA (made up to total with Control siRNA) for 72 hours. The effect of each condition on AR-FL, AR-V7, KLK2, KLK3, FKBP5, TMPRSS2, C-MYC, CBP and p300 mRNA expression (22Rv1 a; LNCaP95 d) and AR- FL, AR-V7, C-MYC, KLK3, CBP, p300 and GAPDH protein expression was determined (22Rv1 b; LNCaP95 e). Mean mRNA expression (normalized to an average of B2M/GAPDH/HPRT1 and control siRNA; defined as 1.0) with standard error of mean from three individual experiments is shown. Growth assays by CellTitre-Glo were carried out (22Rv1 c; LNCaP95 f) (normalized to control siRNA; defined as 1.0) with standard deviation of an individual experiment with six replicates is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for each condition compared to control siRNA (at equivalent concentration) using unpaired student t-test. Western blots are shown in triplicate. (g-i) 22Rv1 cells with doxycycline-inducible short hairpin (sh) RNA to Control (shCON), CBP (shCBP) or p300 (shp300) were treated with 10ng/μg doxycycline for 48 hours prior to transfection with empty vector plasmid (EV), CBP plasmid (CBP rescue) or p300 plasmid (p300 rescue). The effect of each condition on CBP, p300, AR-FL, AR-V7, KLK3, FKBP5, TMPRSS2, C-MYC, ODC and CAD mRNA expression (g) and CBP, p300, AR-FL, AR-V7, C-MYC and GAPDH protein expression (h) was determined after 48 hours. Mean mRNA expression (normalized to 18S and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for shCBP:EV compared to shCBP:CBP rescue, and shp300:EV compared to shp300:p300 rescue, using unpaired student t-test. Western blots demonstrate single experiment. The effect of each condition on relative growth was determined after 5 days using the PicoGreen dsDNA assay (i). Mean relative growth (normalized to baseline and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for shCBP:EV compared to shCBP:CBP rescue, and shp300:EV compared to shp300:p300 rescue, using unpaired student t-test.