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. 2020 Sep 25;11(9):805. doi: 10.1038/s41419-020-03015-6

Fig. 3. A1874-induced anti-colon cancer cell activity is not solely dependent on BRD4 protein degradation.

Fig. 3

The primary human colon cancer cells, pCan1 and pCan2, were treated with A1874 (100 nM) or the vehicle control (“Veh”, 0.2% of DMSO).Cells were further cultured in complete medium for applied time periods, and then expression of listed proteins (a) and mRNAs (b) were shown. The primary human colon cancer cells, pCan1 and pCan2, were treated with A1874 (100 nM), JQ1 (500 nM), I-BET726 (200 nM), CPI203 (500 nM) or the vehicle control (“Veh,” 0.2% of DMSO) and further cultured in complete medium for applied time periods, and then cell viability and apoptosis were tested by CCK-8 (c) and nuclear TUNEL staining (d) assays, respectively. Stable pCan1 cells with the mutant BRD4 expression construct [“BRD4 (Mut)”] or the empty vector (“Vec”) were treated with or without A1874 (100 nM). Cells were then further cultured in complete medium for applied time periods, and expression of listed proteins was shown (e); Cell death and apoptosis were tested by Trypan blue staining (f) and nuclear TUNEL staining (g) assays, respectively. The stable pCan1 cells with CRISPR/Cas9-BRD4-KO-GFP construct (“ko-BRD4” cells) were treated with or without A1874 (100 nM). The control cells with CRISPR/Cas9 empty vector (“Cas9-C”) were left untreated; Cells were further cultured in complete medium for applied time periods, and then expression of listed proteins was shown (h); Cell death and apoptosis were tested by Trypan blue staining (i) and nuclear TUNEL staining (j) assays, respectively. Expression of listed proteins was quantified and normalized to the loading control (a, e, h). Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “Veh” cells. #P < 0.05 vs. A1874 treatment (c, d). #P < 0.05 vs. “Vec” cells (f, g). #P < 0.05 (I and J). Experiments in this figure were repeated three times, and similar results were obtained.