Fig 1: Mechanism of Action of a JQ1-VHL PROTAC(A) Schematic representation of the mechanism of action of a PROTAC.(B) Scatterplots of protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 6 hr with the BET inhibitor JQ1-Az (10 µM), the JQ1-VHL PROTAC (1 and 10 µM), or VHL alkyne (10 µM) relative to vehicle control in two biological replicate experiments. Red closed circles indicate statistically significant regulation (p < 0.05), and BRD2, 3, and 4 and FYTTD1 are labeled exemplarily. The dashed diagonal indicates the equality line. Upper panels indicate regulation of proteins that were already present before compounds were added (mature proteins), and lower panels indicate proteins synthesized after compounds were added (nascent proteins). N indicates the number of proteins robustly quantified in both replicates.(C) Line charts with markers of protein fold changes observed in mature (upper panel) and nascent (lower panel) forms of selected proteins after 6- (left) and 24-hr (right) treatment with JQ1-Az (10 µM, red), JQ1-VHL PROTAC (1 µM, blue; 10 µM, green), and VHL alkyne (10 µM, purple) relative to vehicle control. Triangles and circles distinguish data from biological replicates 1 and 2. Proteins where fold changes measured for both replicates are outside the dotted lines are significantly regulated (p < 0.05).(D) Bar chart indicating relative contributions of nascent (green) and mature (orange) proteins to proteome composition after 6- or 24-hr incubation with vehicle, JQ1-Az (10 µM), and JQ1-VHL PROTAC (1 and 10 µM). Percentages of mature and nascent proteins are estimated from the summed-up reporter ion abundances.
Fig 2: Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC, Related to Figure 3(A) Fluorescence in situ hybridization (FISH) of polyA+RNA detected by a Cy3-labeled oligo-dT50 probe. THP-1 cells were treated with either vehicle, JQ1-Az (10μM), JQ1-VHL-PROTAC (at 1 or 10 μM) or I-BET-151-VHL-PROTAC (10 μM) for 6 hr (3 independent experiments are displayed). Cellular localization of polyA+RNA was visualized with confocal microscopy. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray) are shown. Lower panel displays an overlay of Cy3 staining (gray) and Hoechst nuclear staining (cyan) Scale bar, 20 μm.(B) Ratio of mean fluorescence intensity of FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders were defined by WGA staining, not shown) was calculated for single cells using the CellProfiler software (520-790 cells for experiment 1, 546-791 cells for experiment 2 and 278-578 cells for experiment 3 were quantified per condition) from experiment displayed in (A). SEM is shown.(C) Schematic representation of the TREX complex components. Protein names in red text are found significantly regulated compared to vehicle control upon treatment with JQ1-VHL PROTAC.(D) Thermal proteome profiling (TPP) experiment in HepG2 cells after incubation with 15 μM JQ1 (Tm difference in degree Celsius of JQ1 versus vehicle). Proteins with strongest stabilization in two independent replicates are highlighted. Red color indicates significant changes(E) Thermal stabilization of BRD4, SOAT1 and CYP4F12 induced by 15 μM JQ1 in HepG2 cells displayed as normalized non-denatured protein fraction.(F) 2D-thermal proteome profiling of JQ1 in THP1 cell lysate. Dose-dependent stabilization of BRD2, 3, 4 and SOAT1 are displayed (normalized apparent stability and pEC50 are reported)(G) Thermal shift assay of recombinant FYTTD1 with JQ1.
Supplier Page from Abcam for Recombinant Human FYTTD1 protein