IDR-Targeting Compounds Suppress HPV Genome Replication via Disruption of Phospho-BRD4 Association with DNA Damage Response Factors

Published: 17 October 2023| Version 1 | DOI: 10.17632/bdtkms7by4.1
Shwu-Yuan Wu


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.


University of Texas Southwestern Medical Center at Dallas


Viral Replication, Chemical Compound, DNA Damage Response, Human Papillomavirus Related Disease


National Institutes of Health


Cancer Prevention and Research Institute of Texas