nb100 182ss  (Bethyl)

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: (A) Quantification of chromosomal aberrations in HCT116 parental and ZGRF1 −/− cells. Means with 95% confidence intervals are plotted. p values were calculated using Mann-Whitney U tests. N ≥ 32 spreads per condition. (B and C) Examples of metaphase spreads from parental cells (B) or ZGRF1 −/− cells (C) untreated or treated with 20 ng/mL MMC for 24 h. White arrows mark chromosomal aberrations. The images shown highlight the types of aberrations scored rather than being representative of the number of aberrations seen per spread. (D) ZGRF1 −/− cells show a higher frequency of co-localizing γ-H2AX and FANCD2 foci under both unperturbed conditions and when treated with MMC. Quantification of co-localizing γ-H2AX and FANCD2 foci under unperturbed conditions and with two different treatments with MMC is shown. Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N > 100 cells for each condition. (E) Representative images of HCT116 parental and ZGRF −/− cells under unperturbed conditions or after treatment with 20 ng/mL MMC for 4 h. Arrows mark co-localizing foci for one cell, where the pixel intensity of FANCD2 foci was a minimum of 6,000 arbitrary units higher than background. Cells with very low or absent γ-H2AX foci were not scored, as this indicated inadequate immunostaining. (F) Colony formation assay of HCT116 parental and ZGRF1 −/− and FANCM −/− single- and double-mutant cell lines treated with the indicated doses of MMC for 24 h. The graph of the parental cell line is statistically different from each of the knockout cell lines (p < 0.05, t test), but the knockout cell lines are not significantly different from each other (n.s.). (G) Colony formation assay of HCT116 parental and ZGRF1 −/− and FANCJ single- and double-mutant cell lines treated with the indicated doses of MMC for 24 h. The graph of the parental cell line is statistically different from the ZGRF1-knockout cell line (p < 0.05, t test). The FANCJ single- and double-mutant cell lines are not significantly different from each other (n.s.). Graphs in (F) and (G) show the mean with 95% confidence interval. Statistical significance was calculated using unpaired t tests without assuming consistent SD. *p < 0.05. n.s., no significant difference. n ≥ 3 for each cell line.

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: MANN-WHITNEY, Immunostaining, Colony Assay, Mutagenesis, Knock-Out

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: (A) ZGRF1 localizes to nuclear foci during ICL repair. Cells expressing ZGRF1–2xYFP from the endogenous promoter were synchronized at the G1/S border by treatment with 2 mM thymidine for 18 h before release into S phase in Leibovitz’s L-15 medium containing 0.4 μM Hoechst 33258. Four hours before release, 20 ng/mL of MMC or vehicle was added to the cultures. Arrows indicate ZGRF1 foci. (B) Quantification of ZGRF1 foci after MMC treatment. Quantification of the experiment in (A). Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N = 90–160 cells for each condition. (C) Colocalization of ZGRF1 and FANCD2. Cells expressing ZGRF1–2xYFP from the endogenous promoter and ectopically integrated mCherry-FANCD2 were synchronized in S phase with 2 mM thymidine 18 h prior to microscopy. Four hours before microscopy, 20 ng/mL MMC or vehicle was added to the culture. Yellow arrows mark ZGRF1 foci, red arrows mark FANCD2 foci, and orange arrows mark the co-localizing foci. (D) Quantification of co-localizing FANCD2 and ZGRF1 foci in the experiment reported in (C). Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N > 400 cells for each condition.

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: Expressing, MANN-WHITNEY, Microscopy

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: Imaging, Western Blot, Virus, Recombinant, Protease Inhibitor, In Situ, Plasmid Preparation, Software

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: Imaging, Western Blot, Virus, Recombinant, Protease Inhibitor, In Situ, Plasmid Preparation, Software