Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Expressing, Transfection, Western Blot, Positive Control, Control

Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Comparison, Knockdown, shRNA, Construct, Western Blot

Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Expressing, Transfection, Clone Assay, Western Blot, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 1. DNA damage induces an increase in nuclear levels of Rad51 in Brca2-proficient and Brca2-deficient cells. HeLa (A), HCT116 (B), and Capan-1 (C) cells grown at 37 °C were harvested at the indicated times following exposure to 2 Gy of IR and fractionated as described under “Experimental Procedures” to yield cytoplasmic (Cyto), nucleoplasmic (Nuc), and chromatin (Chrom) samples. D and E, HCT116 and Capan-1 cells, respectively, were treated with cyclo- heximide (CHX; 20 M) 1 h prior to exposure to 2 Gy of IR. A portion of each fraction (30 g of total protein) was loaded onto 4–12% SDS-polyacrylamide gels, andWesternblotsweredevelopedusingamouseanti-Rad51monoclonalantibody.Blotswerealsodevelopedusingthefollowingmarkersasloadingcontrols: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cytoplasmic), Sam68 (nucleoplasmic), and fibrillarin (chromatin). F, changes in levels of nuclear Rad51 as a function of time after IR treatment in A–E were quantified as described under “Experimental Procedures.” The data shown are representative of the results of at least three separate experiments, and the S.D. observed with quantification was 20%.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 2. Levels of Capan-1 nuclear Rad51 increase in an IR dose-de- pendent manner. Capan-1 cells exposed to 5 or 8 Gy of IR were grown at 37 °C for 2 h. Cells were harvested and fractionated as described under “Experimental Procedures,” and portions of the cytoplasmic (Cyto) and nucle- oplasmic (Nuc) fractions (25 g of total protein) were loaded onto 4–12% SDS-polyacrylamide gels. Western blots were developed using a mouse anti- Rad51 monoclonal antibody, and levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures” (supplemen- tal Table 1). The blot shown is representative of four separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 4. Rad51C depletion decreases the steady-state level of nuclear Rad51 and diminishes its DNA damage-inducednucleartransportinBrca2-proficientandBrca2-deficientcells.HeLa(A)andCapan-1(B) cells were transfected with a nonspecific () or Rad51C-specific () siRNA pool (SMARTpool), grown for 42 h at 37 °C, exposed to 6 Gy of IR, and harvested 2 h later. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were analyzed by Western blotting using an anti-Rad51 monoclonal antibody. Changes in levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures.” GAPDH, glyceraldehyde-3- phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Transfection, Western Blot

Journal: Advanced Science

Article Title: Homoisoflavanone Delays Colorectal Cancer Progression via DNA Damage‐Induced Mitochondrial Apoptosis and Parthanatos‐Like Cell Death

doi: 10.1002/advs.202511406

Figure Lengend Snippet: HIF induces DNA damage‐mediated parthanatos in CRC cells. (A) Heatmap of differentially expressed genes in HCT15 and HCT116 cells treated with or without HIF. (B) Venn diagram illustrating overlapping differentially expressed genes between HCT15 and HCT116 cells. (C) GO enrichment analysis of HIF‐regulated genes in HCT15 and HCT116 cells. (D, E) GSEA of genes modulated by HIF in HCT15 and HCT116 cells. (F) Immunofluorescence analysis of γ‐H2AX expression in HCT15 and HCT116 cells following HIF treatment. (G) Expression levels of DNA damage markers (P53, P21, γ‐H2AX, c‐PARP, and PARP). (H) Gel electrophoresis analysis of plasmid DNA treated with HIF. The values represent the mass ratio of PKF to plasmid DNA, indicating that 200 ng of plasmid DNA was incubated with 0, 1, 3, 10, 30, and 100 µg of HIF for 2 h. (I, J) Cell viability assessment of HCT15 and HCT116 cells with HIF or combined with Olaparib (Ola) treatment. (K) Genomic DNA integrity analysis via gel electrophoresis after HIF treatment. (N, O) ROS levels after HIF treatment in HCT15 and HCT116 cells. (L) Time‐course analysis of DNA damage repair‐related proteins, including RAD51 and phosphorylated ATM (p‐ATM), ATR (p‐ATR), and Chk1 (p‐Chk1) in HCT15 and HCT116 cells at 0.5, 2, and 6 h following HIF treatment. ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: The membranes were then blocked with a 5% non‐fat milk TBST (TBS containing 0.1% Tween‐20) for 1 h at room temperature and incubated at 4°C for overnight with primary antibodies against the following proteins: cleaved Caspase 3 (25128‐1‐AP, Proteintech), cleaved Caspase 8 (9496, CST), PARP (9542, CST), cleaved PARP (9541, CST), P21 (2947, CST), P53 (2527, CST), Cyclin A2 (91500, CST), Cyclin D1 (2978, CST), CDK1 (bs‐1341R, Bioss), CDK2 (bs‐10726R, Bioss), CDK7 (bs‐0569R, Bioss), AKT (4691, CST), p‐AKT (4060, CST), ERK (9102, CST), p‐ERK (9101, CST), MEK (9126, CST), p‐MEK (9154, CST), ATR (13934, CST), p‐ATR (2853, CST), Chk1 (2360, CST), p‐Chk1 (2348, CST), RAD51 (14961‐1‐AP, Proteintech), ATM (27156‐1‐AP, Proteintech), p‐ATM (5883, CST), γ‐H2AX (9718, CST), AIF (5318, CST), MIF (75038, CST), Cyt c (10993‐1‐AP, Proteintech), Lamin B (66095‐1‐Ig, Proteintech), VDAC1 (81538‐1‐RR, Proteintech) and GAPDH (60004‐1‐Ig, Proteintech).

Techniques: Immunofluorescence, Expressing, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation