Figure 3.

Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC

(A) Imaging of nuclear RNA content by fluorescence in situ hybridization (FISH) and confocal microscopy. THP-1 cells were treated with vehicle, JQ1-Az (10 μM), JQ1-VHL-PROTAC (at 1 and 10 μM), or I-BET-151-VHL-PROTAC (10 μM) for 6 hr, fixed, and processed for FISH using Cy3-labeled oligo-dT50. Nuclei were stained by Hoechst. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray, upper panel) are shown. The lower panel displays an overlay of Cy3 staining (gray) and Hoechst staining (cyan). Scale bar, 20 μm.

(B) Bar chart displaying the ratio of mean fluorescence intensity of the FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders as defined by WGA staining) was calculated for single cells (706–1,215 cells per condition). Mean fluorescence of treated samples is normalized to control vehicle. SEM is shown. The experiment was repeated three times (Figures S3A and S3B).

(C) Scheme of 2D thermal proteome profiling (2D-TPP) experiments.

(D) 2D-TPP results for JQ1 and I-BET151. Sigmoidal curves show dose-dependent changes in thermal stability for selected proteins. pEC₅₀ is defined as – log₁₀(EC₅₀).

(E) Dose-dependent effects of cellular JQ1 treatment on the thermal stability of five proteins involved in cholesterol biosynthesis revealed by 2D-TPP. The table shows pEC₅₀s for dose-dependent stabilization; the pathway is displayed in the center, and enzymes are marked in blue. Curves depict dose-dependent stabilization in JQ1-treated cells for indicated enzymes.