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: Stem Cell Reports

Article Title: Endogenous DNA Damage Leads to p53-Independent Deficits in Replicative Fitness in Fetal Murine Fancd2 − / − Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.stemcr.2016.09.005

Figure Lengend Snippet: DNA-Damage Responses Are Induced, and the Strand Breaks Are Accumulated in FA FL HSPCs (A) Representative γH2AX foci in SCA1 + FL cells (60× objective lens; the scale bar represents 5 μm). (B) Quantification of γH2AX foci per cell (n = 3 WT and 3 KO from three litters). (C) Representative RAD51 foci in SCA1 + FL (60× objective lens; the scale bar represents 5 μm). (D) Quantification of cells positive for foci (p = 0.02, n = 6 WT and 6 KO from three litters). (E) Expression of DDR genes in SCA1 + FL cells (p = 0.08, 0.44, 0.06, 0.22, and 0.005, respectively; n = 4 WT and 4 KO from three litters). (F) Expression of selected DDR genes in ASL-sorted FL cells (p = 0.08, 0.5, 0.04 respectively; n = 3 WT and 3 KO from two litters). (G) Olive tail moment of 428 SCA1 + FL cells from seven WT animals and 288 SCA1 + FL cells from five Fancc − / − animals from four litters (p = 0.003). (H) Olive tail moment of 289 SCA1 + FL cells from five WT animals and 267 SCA1 + FL cells from five Fancd2 − / − animals from four litters (p = 0.04). (I) Representative alkaline comets of SCA1 + FL cells (20× objective lens; the scale bar represents 20 μm). Error bars reflect SEM, and asterisks indicate ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01.

Article Snippet: Cells were permeabilized with NET buffer with 0.5% Triton X-100 ( ) and stained with Hoechst (Thermo Scientific 62249) and AF488-conjugated anti-γH2AX (BioLegend, 613407), rabbit anti-mouse RAD51 (Santa Cruz Biotechnology, 8349) followed by Cy3 goat anti-rabbit IgG (Millipore, AP132C) or Cy5-conjugated anti-phospho-p38 (T180/Y182) (eBioscience, 17-9078-42).

Techniques: Expressing

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