Journal: bioRxiv
Article Title: Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways
doi: 10.1101/2024.12.27.630542
Figure Lengend Snippet: A, DSB/DDR induction by bleomycin is measured by γ-H2A.X and p-ATM immunoblotting. B, Quantification of γH2A.X foci. Representative images (top panels) and 3D reconstruction (bottom panels, Imaris) of control and HD ISPNs stained with γ-H2A.X specific antibody upon induction of DSB by bleomycin, (10μg/ml, 30min), or from untreated cells (NT). C, graphs show 3D quantification. Data represent mean ±SEM. Two-sample t-tests with equal variances were performed. * p<0.05, n=5. DSB/DDR quantification shows lower γH2A.X foci size and intensity in HD ISPNs compared to normal ISPNs. D, HD ISPNs are more vulnerable to DSB-induced stress. Control (33CAG) and HD (180CAG) ISPNs were treated with 10μg/ml of bleomycin for indicated time and ATP and Casp3/7 levels were measured. Data is presented as mean % ± SEM of corresponding non-treated group. One-way ANOVA with Pairwise Multiple Comparison Procedures (Holm-Sidak method): *p=0.029, n=3; **p=0.001, n=3. T-test with equal variances: ^ p<0.001, n=3; ^^ p=0.004, n=3; ^^^ p=0.017, n=3
Article Snippet: Other antibodies used: ATM [2C1] from Genetex (#GTX70103); phospho-ATM (Ser1981) from R&D Systems (#AF1655); phospho-histone H2A.X (S139) [3F2] from Genetex (#GTX80694); histone H2A.X (D17A3) from Cell Signaling Technology (#7631); phospho DNA-PKcs (S2056) from Abcam (#18192); DNA-PKcs [E6U3A] from Cell Signaling Technology (#38168); TCERG1/CA150 from Bethyl Laboratories (#A300-360A); BRG1[BLR106H] from Bethyl Laboratories (#A700-106); ARID1A/BAF250 [BLR279L] from Bethyl Laboratories (#A700-279); b-actin [C4] from Santa Cruz Biothechnology (#sc-47778).
Techniques: Western Blot, Control, Staining, Comparison