Fig. 4: BET protein inhibitors revert etoposide resistance through SatIII regulation.

A Representative images of co-localization of SatIII RNA and BRD4 after exposure to HS conditions (1 h at 44 °C). Immediately following HS, cells were fixed, immunostained, and imaged. SatIII was stained with smFISH (red), BRD4 with a protein-specific antibody (green). Scale bar, 10 µm. B Binding of BRD4 to the SatIII DNA locus after HS (1 h at 44 °C). For the ChIP-Seq experiment cells were subjected to HS or control conditions. ChIP was performed with a BRD4-specific antibody. C Effect of BRD4 inhibition on SatIII expression. HeLa cells were treated with various BRD4 inhibitors and exposed to HS conditions (1 h at 44 °C). Two read-out methods were applied: RNA expression was measured by qPCR and RNA FISH was used to quantify SatIII RNA foci. Values of non-treated cells (DMSO control) were set to 100%. Error bars represent SD of the mean of n = 2 independent replicates. P-values < 0.05 marked with (*). Significance was determined using two-tailed paired Student’s t-test. D Cell viability of HeLa cells treated with etoposide and the BRD4 inhibitor JQ1 (1 µM) or DMSO as control. Directly after exposure to HS (1 h at 44 °C), cells were treated with the indicated etoposide concentrations in combination with JQ1. After an additional 48 h, cell viabilities were measured using AlamarBlue. Error bars represent SD of the mean of n = 2 replicates. P-values <0.05 are marked with (*), Significance was determined using two-tailed unpaired Student’s t-test. E Same experiment as in (D) but with the BRD4 inhibitor CPI203 (1 µM). F Cell proliferation of HeLa cells either stably overexpressing SatIII RNA or an empty vector control. Cells were treated with 20 µM etoposide and cell proliferation was measured by acquisition of images every 30 min over a time course of 48 h. Confluency was analyzed with the cell profiler software. G Cell proliferation assay performed as described in (F) but with a combination treatment of 20 µM etoposide and 5 µM JQ1.