Fig. 4.

(A) Illustration of intracellular hypoxic enzyme uncaging, formation of the degrader (click J252), and degradation of BRD4. (B) Western blot analysis of BRD4 protein levels after treatments with GSH-cleavable J266 (6 hours) and JQ1-CBT (12 hours) at indicated concentration and different time points; GSH- and NTR-responsive JW4 (6 hours) with JQ1-CBT (12 hours) at the indicated concentration and for a different time period under hypoxia in HEK-293T cells. (C) Analysis of BRD4 protein levels under the combination of different control conditions for 12 hours (the inhibitor bortezomib incubated with cells for 2 hours before the addition of JW4 and JQ1-CBT). (D) Western blot analysis of BRD4 protein levels after treatments with J266 (6 hours) and JQ1-CBT (12 hours) or JW4 (6 hours) and JQ1-CBT (12 hours) in time- and concentration-dependent examinations of HeLa cancer cells; analysis of BRD4 protein levels after treatments with JQ1 (12 hours) in a concentration-dependent manner of hypoxia HeLa cells; and JW4 (6 hours) and JQ1-CBT (12 hours) in normoxia HeLa cells. (E) Extended concentration of degraders demonstrating the hook effect during incubation under hypoxia (representative results from two to three biological replications). β-Tubulin was used as internal controls. (F) BRD4 degradation level over varied concentration as indicated in (E). Values represent triplicate means ± SD, normalized to nontreated cells, and baseline-corrected using immunoblots.