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:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Immunocytochemical staining for FANCD2. A: Fibroadenoma showing strong (+++) nuclear staining (N) and moderate (++) cytoplasmic staining (C) of the tumor cells. B: Fibroadenoma at higher magnification showing stronger nuclear staining of the myoepithelial cells (M) than the surrounding epithelial cells (E) of the tumor. C and D: Invasive carcinoma showing strong (+++) cytoplasmic staining (arrows) of the carcinoma cells at lower (C) and higher (D) magnification. E: invasive carcinoma showing no nuclear (N) or cytoplasmic staining (C) of the carcinoma cells. F: invasive carcinoma showing borderline (1–5%) cytoplasmic staining (arrow) of the carcinoma cells. G and H: Serial sections of invasive carcinoma incubated with (G) anti-FANCD2 and (H) anti-FANCD2 pre-incubated with immunizing peptide showing (G) strong (+++) cytoplasmic staining (arrows) and (H) no staining (−), respectively. Magnification: ×220 A and C; ×550 B and D–H. Scale bar: 50 μm (A and C); 20 μm (B and D–H).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining, Incubation

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Nuclear and Cytoplasmic Staining for FANCD2 in Both Benign and Malignant Lesions

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Nuclear and Cytoplasmic Staining of Human Cell Lines for FANCD2

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Immunoblotting for FANCD2 in nuclear and cytoplasmic fractions of cultured cell lines. A: Lysates of breast cell lines SVE3, Huma 121, MCF-7, and MDA-MB-157 were fractionated into nuclear and cytoplasmic extracts and Western blotted for FANCD2 (using H300 anti-rabbit antibody). MMC treatment (+MMC) was at 50 nmol/L for 18 hours before the preparation of lysates. Nuclear (N) extracts show the presence of TATA binding protein (TBP) while cytoplasmic (C) extracts show the presence of tubulin, with no detectable cross-contamination. Immortalized normal (SVE3) and immortalized benign (Huma 121) cell lines both express immunodetectable FANCD2-S (non-monoubiquitylated; 155 kDa) and FANCD2-L (monoubiquitylated; 162 kDa) in the nucleus (irrespective of MMC-treatment), while only FANCD2-S is detectable in the cytoplasm. In the low invasive malignant cell line MCF-7, only the non-ubiquitylated FANCD2 (155 kDa) is immunodetectable and this is confined to the cytoplasm of these cells. The high invasive malignant cell line MDA-MB-157 exhibits no immunodetectable FANCD2 in either fraction. The arrows indicate the position of the marker proteins for FANCD2-L, FANCD2-S, tubulin, and TBP. B: PD20 cells (FA-D2 complementation group) and PD20-3-15 cells (PD20 functionally complemented with human chromosome 3p) were treated with 50 nmol/L MMC for 18 hours before the preparation of lysates. PD20 exhibits no immunodetectable FANCD2 protein. In PD20-3-15, both FANCD2-S (non-monoubiquitylated; 155 kDa) and FANCD2-L (monoubiquitylated; 162 kDa) are present in the nucleus, while only FANCD2-S is detectable in the cytoplasm.

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Western Blot, Cell Culture, Binding Assay, Marker

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Fanconi anemia-like phenotype in malignant breast cancer cell lines that lack FANCD2 expression. A: Western blots of high invasive malignant cell lines MDA-MB-157 and MDA-MB-231. They lack immunodetectable FANCD2 in whole cell extracts (cells treated with 50 nmol/L MMC for 18 hours before preparation of lysates). Transduction of a human cDNA for FANCD2 into MDA-MB-157 restores the expression of both isoforms of FANCD2. Loading has been normalized to that of β-actin and the arrows indicate the position of the marker proteins for FANCD2-L, FANCD2-S, and β-actin. B: Growth inhibition by MMC is shown as the mean of three independent experiments for each of the indicated cell lines. The mean percentages (±SD) of cells after exposure to MMC for 72 hours, relative to the untreated cells are shown. MDA-MB-157 and MB-MB-231 exhibit a response to MMC that is similar to that shown by the Fanconi anemia patient FA-D2 cell line PD20. The MMC-response of MDA-MB-157 cells transduced with an expression vector for FANCD2 (+FANCD2), is very similar to PD20-3-15, indicating functional complementation of MDA-MB-157 by the wild-type FANCD2 cDNA. C: Induction of chromosomal aberrations in MDA-MB-157 and MDA-MB-157+FANCD2 by MMC. Chromosomal aberrations were scored by metaphase analysis in cells exposed to MMC for 24 hours before the addition of colcemid for 2 hours. For each cell line/treatment, 20 metaphases were scored for chromosomal aberrations using the criteria described by Scott et al.35 Chromatid breaks or exchanges involve single arms of metaphase chromosomes, while chromosome breaks or exchanges involve both arms. No chromosomal aberrations were observed in untreated MDA-MB-157 cells while 2 chromosome exchanges (a ring chromosome and a dicentric) were observed in untreated MDA-MB-157+FANCD2 cells. On treatment with MMC, the level of aberrations in MDA-MB-157 increased to a mean of 3.5 chromosomal aberrations per cell. On average, 2.1 chromatid breaks, 1.3 chromatid exchanges and 0.1 chromosome breaks per cell were observed, with all of the chromatid exchanges being complex arrangements (exchanges involving more than two chromosomes), typical of those seen in FA patient cells following MMC treatment.44 In contrast, MDA-MB-157+FANCD2 cells exhibited a much more modest increase in chromosomal aberrations with MMC (0.4 per cell; 5 chromatid breaks, 2 chromatid exchanges and a single chromosome exchange in the 20 metaphases scored).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Expressing, Western Blot, Transduction, Marker, Inhibition, Plasmid Preparation, Functional Assay

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Association of Immunocytochemical Staining for Cytoplasmic FANCD2 with Other Tumor Variables

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Association of immunocytochemical staining of the primary carcinoma for cytoplasmic FANCD2 with overall survival of patients. A: The cumulative proportion of patients surviving as a fraction of the total at 12 monthly intervals after presentation for patients with carcinomas classified as follows: (a) +++, strong staining (——– ; 100% = 5 patients); (b) ++, moderate staining (------; 100% = 36 patients); (c) +, intermediate staining (........ ; 100% = 55 patients); (d) +/−, borderline staining (– – –); 100% = 96 patients) and (e) −, negative staining (- — - —, 100% = 118 patients) for FANCD2. There were 5 censored observations in a (4 dead of other causes (doc); cumulative proportion surviving (cps) = 100%; median survival time (mst) > 204 months); 31 in b (14 doc; cps = 84%; mst >216 months); 52 in c (13 doc; cps = 94%; mst >216 months); 65 in d (17 doc; cps = 64%; mst >228 months); and 15 in e (12 doc; cps = 4%; mst = 46.8 months). The cumulative proportions surviving ± 95% CIs were (a) 1.00 ± 0.0, (b) 0.91 ± 0.05, (c) 0.94 ± 0.03, (d) 0.75 ± 0.05, and (e) 0.33 ± 0.04, after 5 years; (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.69 ± 0.05, and (e) 0.16 ± 0.03, after 10 years; (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.64 ± 0.05, and (e) 0.04 ± 0.03, after 15 years; and (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.64 ± 0.05, and (e) 0.04 ± 0.03, after 20 years. The five curves were significantly different (Wilcoxon statistic χ2 = 125.83, 4 d.f., P < 0.0001), and significantly different in the successive pairwise combinations of c with d (χ2 = 13.85, 1 d.f., P < 0.0001) and d with e (χ2 = 53.29, 1 d.f., P < 0.0001) but not b with c (χ2 = 2.03, 1 d.f., P = 0.155) nor a with b (χ2 = 0.63, 1 d.f., P = 0.43). The malignant grade of the 5 strong staining carcinomas (+++) (a) was confirmed as grade I (2 patients), II (2 patients), and III (1 patient). B: The cumulative proportion of patients surviving as a fraction of the total at 12 month intervals after presentation with carcinomas classified as (a) positive staining using a 1% cut off (——– ; 100% = 192 patients) and (b) −, negative staining (----- ; 100% = 118 patients) for FANCD2. There were 153 censored observations in a (48 doc; cps = 77%; mst >228 months) and 15 in b (12 doc; cps = 4%; mst = 46.8 months). The cumulative proportions surviving ± 95% CIs were (a) 0.84 ± 0.03, (b) 0.33 ± 0.04, after 5 years; (a) 0.80 ± 0.03, (b) 0.16 ± 0.03, after 10 years; (a) 0.77 ± 0.03, (b) 0.04 ± 0.03, after 15 years; and (a) 0.77 ± 0.03, (b) 0.04 ± 0.03, after 20 years. The two lines were significantly different (Wilcoxon statistic χ2 = 117.14, 1 d.f., P < 0.0001).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining, Negative Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Summary of the Cox’s Proportional Hazards Model for Cancer-Related Deaths

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques:

Journal: British Journal of Cancer

Article Title: Tumour regression and ERCC1 nuclear protein expression predict clinical outcome in patients with gastro-oesophageal cancer treated with neoadjuvant chemotherapy

doi: 10.1038/sj.bjc.6605686

Figure Lengend Snippet: Nuclear expression and correlation with lack of tumour response (TRG 4 or 5) in the neoadjuvant chemotherapy group

Article Snippet: For FANCD2 analysis the slides were incubated for 1 h with the primary anti-FANCD2 antibody (Novus Biologicals Inc., Littleton, CO, USA) in a dilution of 1 : 200.

Techniques: Expressing

Journal: bioRxiv

Article Title: Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

doi: 10.1101/2025.01.06.631600

Figure Lengend Snippet: Top: (A) Locus map of a 296 kb region in the CFS-NFIA obtained by SbfI digestion. The FISH probes that identify the segment are labeled in blue. Top; Locus map of the SbfI digested NFIA segment. Bottom; Aligned photomicrograph images of labeled DNA molecules from DNA Replication program at CFS-NFIA in IMR90 fibroblasts (B), FANCD2 −/− fibroblasts(C) and XPV −/− fibroblasts(D). The yellow arrows indicate the sites along the molecules where the IdU transitioned to CldU. The yellow arrows with white ovals indicate replication fork origin activation. The molecules are arranged in the following order: molecules with initiation events, molecules with 3’-to-5’ progressing forks, molecules with 5’-to-3’ progressing forks, and molecules with termination events. (E) The percentage of molecules incorporating IdU (red) is calculated from the replication program (middle) and is represented as a histogram. (F) The percentage of molecules with replication forks at each 10 kb interval of NFIA. Black arrows denote the most prominent pause peaks and correspond to the white ovals in the SMARD profile.

Article Snippet: Primary Antibodies for western blot included anti-DNA Polymerase Eta (Cell Signaling-13848S, 1:2000), anti-yH2AX (Cell Signaling – 9718T), anti-pCHK1 (s317) (Cell signaling –123402), anti-H3 (Cell Signaling – 12648T), anti-PCNA mAB (Cell Signaling-2586T, 1:1000), anti-ubiquityl PCNA mAB (Cell Signaling-13439S, 1:1000), anti-FANCD2 (SantaCruz, 1:2500), phospho-ATR (Cell Signaling, 30632, 1:2000)

Techniques: Labeling, Activation Assay

Journal: bioRxiv

Article Title: Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

doi: 10.1101/2025.01.06.631600

Figure Lengend Snippet: (A) Expression levels of ubPCNA and PCNA in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells treated with and without Aphidicolin (APH) using western blotting. H3 is used as loading control. (B) Expression levels of Pol eta in PIP*, UBZ*, F1* and Pol eta +/+ cells treated with and without Aphidicolin (APH) using immunofluorescence staining. ( C-F) Chromatin recruitment of RAD51, BRCA1, BRCA2 and FANCD2 proteins assessed using immunofluorescence staining. Representative images are depicted on the right. (G) FANCD2 mono-ubiquitination determined by western blotting in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells treated with Aphidicolin (APH).

Article Snippet: Primary Antibodies for western blot included anti-DNA Polymerase Eta (Cell Signaling-13848S, 1:2000), anti-yH2AX (Cell Signaling – 9718T), anti-pCHK1 (s317) (Cell signaling –123402), anti-H3 (Cell Signaling – 12648T), anti-PCNA mAB (Cell Signaling-2586T, 1:1000), anti-ubiquityl PCNA mAB (Cell Signaling-13439S, 1:1000), anti-FANCD2 (SantaCruz, 1:2500), phospho-ATR (Cell Signaling, 30632, 1:2000)

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

doi: 10.1101/2025.01.06.631600

Figure Lengend Snippet: (A) Time course experiment to measure recovery of cells after release into drug free media over the course of 48 hours. Cells were collected at six time points (0, 4, 8, 12, 24, 28, 36 and 48 hours) and expression levels of FANCD2 mono-ubiquitination was determined by western blotting. (B) Analysis of number of FANCD2 twin-foci (red) per mitotic cell nucleus (DAPI, blue) in Pol−/−, PIP*, UBZ*, F1* and Pol eta+/+ treated with Aphidicolin (APH), representative images are shown on the bottom, n=100; (C) Analysis of number of EdU foci (green), sandwiched between FANCD2 twin-foci(red), per mitotic cell nucleus (DAPI, blue) in Pol−/−, PIP*, UBZ*, F1* and Pol eta+/+ cells treated with Aphidicolin (APH), representative images are shown on the bottom, n=100. (D) Analysis of number of gH2AX foci (red) in the cytoplasm surrounding each cell nucleus in Pol −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells with or without Aph treatment. Representative images are on the top, n=100; (E) Analysis of number of cGAS (green) in the cytoplasm surrounding each cell nucleus in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells with or without Aph treatment. Representative images are in the middle, n=100. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. (F) Analysis of number of Pico green foci in the cytoplasm surrounding each cell nucleus in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells with or without Aph treatment. Representative images are in the middle, n=100. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001.

Article Snippet: Primary Antibodies for western blot included anti-DNA Polymerase Eta (Cell Signaling-13848S, 1:2000), anti-yH2AX (Cell Signaling – 9718T), anti-pCHK1 (s317) (Cell signaling –123402), anti-H3 (Cell Signaling – 12648T), anti-PCNA mAB (Cell Signaling-2586T, 1:1000), anti-ubiquityl PCNA mAB (Cell Signaling-13439S, 1:1000), anti-FANCD2 (SantaCruz, 1:2500), phospho-ATR (Cell Signaling, 30632, 1:2000)

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

doi: 10.1101/2025.01.06.631600

Figure Lengend Snippet: (A) In non-affected cells, stress induced replication stalling is efficiently resolved leading to CFS stability and over genome stability. (B) Inactivation of the UBZ domain (and to a certain extent the PIP domain) of Polη results in extensive replication stalling, inefficient fork restart, leading to under-replicated DNA in G2/M. However, while the F1* effectively resolved (Genomic stability ) under-replicated DNA in G2/M by MiDAS, the UBZ and PIP*, like pol eta −/− display persistent damage that leads to mis segregated DNA during mitosis ( Genomic instability ) . Importantly, The UBZ* mutant like the pol eta −/− cells showed a prominent presence of chromosomal mis-segregation in the form of FANCD2+ micronuclei.

Article Snippet: Primary Antibodies for western blot included anti-DNA Polymerase Eta (Cell Signaling-13848S, 1:2000), anti-yH2AX (Cell Signaling – 9718T), anti-pCHK1 (s317) (Cell signaling –123402), anti-H3 (Cell Signaling – 12648T), anti-PCNA mAB (Cell Signaling-2586T, 1:1000), anti-ubiquityl PCNA mAB (Cell Signaling-13439S, 1:1000), anti-FANCD2 (SantaCruz, 1:2500), phospho-ATR (Cell Signaling, 30632, 1:2000)

Techniques: Mutagenesis