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. 2021 Mar 25;13(7):1519. doi: 10.3390/cancers13071519

Figure 3.

Figure 3

BET inhibition induces DNA damage and perturbs tumor spheroid architecture. (a) WB analysis (left panel) of γH2AX in SKOV3 cells treated with OTX015 (0–1–3 μM) for 72 h. Tubulin served as internal control. IF experiments (right panel) showing the presence of γH2AX foci (red) in OTX015 treated (1 and 3 μM) cells. DMSO was used in control cells. Nuclei (blue) were visualized with DAPI staining. Representative images captured under ApoTome microscope at 40× magnification. (b) q-PCR analysis of NRF2 mRNA levels (left panel) in SKOV3 cells exposed to OTX015 (0–1–3 μM), graphed as fold increase over not-treated cells, set at 1. β-actin mRNA was used as endogenous control. Histograms indicate mean values ±SD of two independent experiments, each performed in triplicate. Statistical significance vs. control cells was calculated by one-way ANOVA (* p < 0.05). WB experiments (upper right panel) performed on protein extracts from SKOV3 cells treated with OTX015 (0–1–3 μM) for 72 h. Tubulin served as loading control. IF experiments (lower right panel) showing the downregulation of NRF2 protein expression (red) in OTX015-exposed (1 μM and 3 μM) cells respect to untreated SKOV3 samples. (c) Transcript levels of GST-M1 and GST-T1 genes by q-PCR analysis in SKOV3 cells exposed to OTX015 (0–1–3 μM) and graphed as fold increase over not-treated cells, set at 1. β-actin mRNA was used as endogenous control. Histograms indicate mean values ±SD of two independent experiments, each performed in triplicate. Statistical significance vs. control cells was calculated by one-way ANOVA (* p < 0.05). (d) OTX015 alters spheroid formation in SKOV3 cells cultured in 24-well ultralow attachment plates with DMEM-F12/B27/EGF/FGF medium. Cells were photographed under a light microscope at 20× magnification (left panel) after 10 days of 3 μM OTX015 treatment (DMSO as control). WB of CD133 protein levels (right panel) in OTX015-exposed (0–3 μM) spheroids. Tubulin served as loading control. The original Western Blot images can be found in Figure S4.