Fig 2: HPI-1 is a high-affinity BET bromodomain binder that increases cellular BRD2 levels.a Representative micrographs of SHH-GFP cells treated with DMSO or 20 µM of HPI-1 for 27 h. Scalebar 25 µm. b High-content microscopy dose-response curve for cells treated with increasing concentrations of HPI-1 and probed for nuclear BRD2. Representative curve from N = 3 independent experiments, with n = 9 images analyzed per condition. c qPCR analysis of Brd2 mRNA levels from cells treated with 5 µM HPI-1, 1 µM HPP-9 or JQ1 for 27 h in the presence of ShhN. Data from N independent experiments, as indicated in the bars, performed in duplicate. One-way Brown–Forsythe and Welch ANOVA, Dunnett T3 test. d The affinity of HPI-1 and HPP-9 to the indicated bromodomains (BromoKd, Eurofins discovery). (1,2) indicates which of the two bromodomain subunits were included. e Proposed binding mode for HPI-1. The interacting residues and HPI-1 are modeled in stick, while BRD2(2) is shown in cartoon representation. The dashed yellow lines represent H-bonds while the p-p stacking is shown as a purple dashed line. f Possible physical model of the ternary complex. BRD2(2) (green) and CRBN (gray) are shown in a surface representation, while HPP-9 (orange) is shown as spheres. Source data are provided as a Source Data file.

Fig 3: HPP-9 and dBet6 have a different mode of action.SHH-GFP cells were treated with 1 µM of HPP-9 or dBet6 for 27 h and probed for GLI1, BRD2, BRD3, and BRD4 by western blot (a) and analyzed by fluorescence microscopy (b, c). Representative micrographs showing the nuclear localization of GFP, BRD2, BRD3, and BRD4 are shown in (b), and the signal is quantified in (c). Scalebar 25 µm. Data shown is the mean from N independent experiments as indicated in or above the bars. At least seven images were analyzed per condition in each experiment. All compounds were used at 1 µM. Two-way ANOVA. d Full dose-response curves were measured by high-content microscopy to determine the pDC50 values for HPP-9 and dBet6. Each dot is an individual image (n is at least 7 images/experiment, N = 3, 4 independent experiments as indicated in the table under the graphs, n.a. not active). e BRD2 and BRD4 binding to Gli1 and Gli2 loci was measured by ChIP-qPCR using two primer pairs per locus. Representative data of two independent experiments. f Volcano plot showing differentially expressed genes in SHH-GFP cells upon 8 h of HPP-9 treatment (+ShhN) compared to DMSO + ShhN. Two independent experiments, padj < 0.05 and 1.5x fold change. Exact p values can be found in Supplementary Data 3 and were determined using the Wald test. The adjusted p values were corrected for multiple testing, using the Benjamin–Hochberg correction. Source data are provided as a Source Data file.