IDR-Targeting Compounds Suppress HPV Genome Replication via Disruption of Phospho-BRD4 Association with DNA Damage Response Factors
The dataset includes raw images used in the Mol. Cell revision (MOLECULAR-CELL-D-23-00513).
Steps to reproduce
The protocols and reagents used to generate these data can be found in the STAR*METHODS. Briefly, for IF images to detect BrdU and HPV genome in the raft culture, deparaffinized tissue sections were subjected to heat-induced epitope retrieval in 10 mM citrate buffer (pH 6.0) for 10 minutes at sub-boiling temperature (95°C) followed by slow cooling to room temperature. Tissue sections were incubated with mouse anti-BrdU antibody which was then bound by horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Invitrogen) followed by Cyanine 3 (Cy3)-labeled tyramide treatment using Tyramide Signal Amplification (TSA) kit (Akoya Biosciences). For subsequent FISH detection, the tissue sections after three washes in 1x PBS were treated with RNase A (100 Kunitz units/ml) for 1 hour at 37°C, dehydrated through graded ethanol (2 minutes each in 70, 90, and 100% ethanol), dried on a hot plate at 50°C, denatured at 74°C for 2 minutes in 70% formamide/2x SSC (pH 7.2,), quickly chilled in 70% ethanol for 2 minutes, dehydrated by treating with 90% and 100% ethanol for 2 minutes each, and dried on a hot plate at 50°C. Next, biotin-labeled HPV18 DNA probe in Hybrisol VII after denatured at 74°C for 10 min was added to the tissue sections, which were then covered by glass cover slips, sealed by rubber cement (Walmart), and incubated overnight (12–16 h) at 37°C. Tissue sections were then sequentially washed at 37°C with: 1) 50% formamide in 1x SSC (pH 7.2), 3 times for 10 minutes each; 2) 1x SSC, one time for 30 minutes; 3) 3% hydrogen peroxide in 1x SSC, one time for 15 minutes; and 4) 2x SSC, 3 times each 15 minutes. After blocking buffer (1% casein in 4x SSC) treatment for 30 minutes at 37°C, tissue sections in the blocking buffer were incubated at 37°C with 1:100 diluted HRP-labeled streptavidin (TSA-Fluorescein System Kit, Akoya Biosciences) for 1 hour and washed at room temperature sequentially with 4x SSC/0.1% Triton X100 twice for 5 minutes each and 4x SSC once for 5 minutes. For Tyramide signal amplification, rinsed tissue sections were treated with Tyramide-Fluorescein (TSA-Fluorescein System Kit) for 10 minutes. After this step, the tissue sections were again rinsed in 4x SSC/0.1% Triton X100 and 4x SSC as above to remove excess unbound fluorophores. Finally, the tissue sections were mounted with DAPI-containing media (Vectashield, H1200) and stored at -20°C. Images were captured with a 20x objective in an Olympus BX63 microscope fitted with appropriate fluorescence filters (Chroma) using an Olympus DP73 camera and cellSens software. Fluorescence excitation/emission wavelengths are 550/570 nm for Cy3 and 494/517 nm for fluorescein. All images in each set of experiments were collected at the same setting. Images were processed using Adobe Photoshop software. For immunoblotting detection of low-abundant proteins, highly sensitive ECL reagents (e.g., Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate) were used.
National Institutes of Health
Cancer Prevention and Research Institute of Texas