Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT, LIPT1 −/− H460, and LIPT1 −/− H460 reconstituted Myc-LIPT1 cells. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. ( B ) Quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT and LIPT1 −/− H157 cells. ( C and D ) Immunoblotting analysis of ATM-pS1981, Ku70, γH2AX, and histone 3 in the soluble nuclear and chromatin fractions of WT and LIPT1 −/− in H460 (C) and in H157 (D) cells, with or without IR (0.5 hours post 10 Gy). Histone H3 and γH2AX served as chromatin markers. LE, long exposure; SE, short exposure. ( E and F ) Immunofluorescence images and quantification of colocalized ATM-pS1981 (red) and γH2AX (green) foci at 0.5 hours post-4 Gy in WT, LIPT1 −/− H460, and Myc-LIPT1–reconstituted H460 cells (E), as well as WT and LIPT1 −/− H157 cells (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. ( G and H ) Immunoblot analysis of ATM-pS1981, total ATM, Chk2-pT68, total Chk2, lipoyl-DLAT/DLST, and γH2AX in H460 (G) and H157 (H) cells at 0.5 hour post-10Gy. (A), (B), (E), and (F), quantification was performed on >100 cells per treatment. Two-way ANOVA was used for (A) and (B), one-way ANOVA was used for (E), and unpaired t test was used for (F). **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Control, Staining, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative immunoblots validating siRNA suppression of KDM4B in WT and LIPT1 −/− H460 cells, with GAPDH used as a loading control. ( B ) Representative images and quantification of H3K9me3 by immunofluorescence staining in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( C and D ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 interaction (C), and between ATM and TIP60 (D) in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells, with or without 10- and 4-Gy IR. ( E ) Representative images and quantification of γH2AX foci by immunofluorescence staining in nonirradiated cells and at 6 and 24 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( F ) Representative images and quantification of RAD51 foci by immunofluorescence staining in nonirradiated cells and 6 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. For (B) to (F), nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: Western Blot, Control, Immunofluorescence, Staining, In Situ, Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 in WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG and 10-Gy IR. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. **** P < 0.0001. ( B ) Immunoblotting analysis of ATM-pS1981 and histone H3 in the chromatin fractions of WT and LIPT1 −/− in H460 cells with or without 1 mM dimethyl–α-KG, 1 mM α-KG, and 10-Gy IR. Histone H3 was used as marker and internal control for the chromatin fraction. Protein levels of ATM-pS1981 were normalized with internal control histone H3. ( C ) Clonogenic assay of WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG after 2, 4, and 6 Gy. The surviving fraction was normalized to the corresponding sham control and survival curves were fitted using the linear-quadratic model. ( D ) Schematic illustrating how LIPT1 deficiency impairs the IR-induced, TIP60-ATM–mediated HR damage repair pathway due to a deficiency in α-KG–dependent demethylation.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Staining, Imaging, Western Blot, Marker, Control, Clonogenic Assay

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