Journal: PLoS ONE

Article Title: The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair

doi: 10.1371/journal.pone.0036970

Figure Lengend Snippet: ( A ) Colony survival of indicated cell lines after exposure to increasing doses of UV light. Error bars represent one standard error of the mean from at least three independent experiments. t -tests have been performed for indicated survival curves. P≥0.05 not significant (NS); ( B ) Exponential growth curve of indicated cell lines. Cell density measurements were carried out over a 72 hr time period. Error bars represent one standard error of the mean from at least two independent experiments that were performed in duplicate. t -tests have been performed for indicated growth curves with calculated P values P≤0.05 (*); ( C ) Immunoblot analysis to monitor MMC-induced FANCD2 monoubiquitylation. Cells were mock treated (−) or exposed to 150 ng/ml MMC (+) for 18 hrs and whole cell lysates were probed for FANCD2 by immunoblotting. Monoubiquitylated FANCD2 resolves as a slower migrating band (indicated by D2-Ub). ( D ) Immunoblot analysis to monitor UV light-induced FANCD2 monoubiquitylation. Cells were irradiated with indicated doses of UV light and allowed to recover for 0, 0.5, 3 and 6 hrs. Whole cell lysates were probed for FANCD2 by immunoblotting.

Article Snippet: The following primary antibodies for immunoblot analyses were used in this study: FANCD2 , pS345Chk1 (Cell Signalling Technology), γ-H2AX (Bethyl), XPC and PCNA (Abcam), and β-actin (Cell Signalling Technology), (6–4)photoproducts (Cosmo Bio Co.), cyclobutane pyrimidine dimers (CPDs) (Sigma).

Techniques: Western Blot, Irradiation

Journal: Genome Medicine

Article Title: A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

doi: 10.1186/s13073-021-00998-5

Figure Lengend Snippet: The isoform with the p.L605F variant impairs FANCI stability and function. a Western blots of HeLa cells with the FANCI gene (FANCI +/+ ) or with the FANCI gene knocked out (FANCI −/− ). HeLa FANCI −/− cells from clone 1 were complemented with constructs of Flag-FANCI wild type (WT), p.L605F or p.P55L, or an empty vector (EV) and treated with 50 ng/ml MMC for 18 h. The upper band, H, shows the ubiquitination of FANCI and FANCD2 after treatment. The lower band, L, corresponds to non-ubiquitinated FANCI or FANCD2. VINCULIN was used as a loading control. Experiment was repeated three times. b HeLa FANCI +/+ cells were transfected with siRNA targeting FANCI and then complemented with Flag-FANCI siRNA-resistant constructs or an EV. Cells were treated with 50 ng/ml MMC for 18 h followed by FLAG immunoprecipitation. The left panel shows FANCI constructs expression and the right panel the immunoprecipitated fractions. The p.L605F immunoprecipitation fraction sample was super-loaded to have the same signal after FANCI WT complementation. The ratio between the upper band (H) and lower band (L) for the immunoprecipitated FANCD2 is shown. c Immunofluorescence of HeLa FANCI −/− cells from clone 1 that were complemented with constructs of Flag-FANCI and 0.1 μg of empty GFP vector was used as a transfection control. The adjacent scatter plot shows the number of FANCD2 foci in GFP-positive cells after treatment with MMC (50 ng/ml, 18 h). Mean with SEM is represented. The Kruskal-Wallis test was used to compare groups and the P value is shown for each test. Experiment has been performed in triplicate. d–f Western blot analysis of HeLa FANCI −/− cells from clone 1 that were complemented with constructs of Flag-FANCI and treated with cycloheximide (CHX) and either mock-treated ( d ) or treated with damaging agents formaldehyde ( e ) or MMC ( f ) for different lengths of time at the indicated concentrations. At each time point, whole cell extracts were analysed by western blot to assess protein levels. Experiment has been done in triplicate. g Survival curves of HeLa FANCI −/− cells from clone 1 that were transfected with the different constructs of Flag-FANCI. Cell viability was monitored following cisplatin or olaparib treatments for 72 h and was assessed by counting remaining nuclei. Curves represent mean with SEM of three biological replicates. Western blots were used to monitor expression and shown here as an example. Alpha-tubulin was used as a loading control. Full blots are shown in Additional file

Article Snippet: The antibodies used were anti-FANCI (A7) (Santa Cruz Biotechnology, ref sc-271316, 1:100 for western blot), anti-FANCD2 (Novus, ref NB100-182D1, 1:5000 for western blot, 1:1000 for immunofluorescence), anti-Flag (Cell signaling Technologies, ref 8146, 1:1600 for immunofluorescence), and anti-vinculin (Sigma, ref V9131, 1:100,000 for western blot).

Techniques: Variant Assay, Western Blot, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Expressing, Immunofluorescence

Journal: Genome Medicine

Article Title: A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

doi: 10.1186/s13073-021-00998-5

Figure Lengend Snippet: The isoform with the p.L605F variant impairs FANCI stability and function. a Western blots of HeLa cells with the FANCI gene (FANCI +/+ ) or with the FANCI gene knocked out (FANCI −/− ). HeLa FANCI −/− cells from clone 1 were complemented with constructs of Flag-FANCI wild type (WT), p.L605F or p.P55L, or an empty vector (EV) and treated with 50 ng/ml MMC for 18 h. The upper band, H, shows the ubiquitination of FANCI and FANCD2 after treatment. The lower band, L, corresponds to non-ubiquitinated FANCI or FANCD2. VINCULIN was used as a loading control. Experiment was repeated three times. b HeLa FANCI +/+ cells were transfected with siRNA targeting FANCI and then complemented with Flag-FANCI siRNA-resistant constructs or an EV. Cells were treated with 50 ng/ml MMC for 18 h followed by FLAG immunoprecipitation. The left panel shows FANCI constructs expression and the right panel the immunoprecipitated fractions. The p.L605F immunoprecipitation fraction sample was super-loaded to have the same signal after FANCI WT complementation. The ratio between the upper band (H) and lower band (L) for the immunoprecipitated FANCD2 is shown. c Immunofluorescence of HeLa FANCI −/− cells from clone 1 that were complemented with constructs of Flag-FANCI and 0.1 μg of empty GFP vector was used as a transfection control. The adjacent scatter plot shows the number of FANCD2 foci in GFP-positive cells after treatment with MMC (50 ng/ml, 18 h). Mean with SEM is represented. The Kruskal-Wallis test was used to compare groups and the P value is shown for each test. Experiment has been performed in triplicate. d–f Western blot analysis of HeLa FANCI −/− cells from clone 1 that were complemented with constructs of Flag-FANCI and treated with cycloheximide (CHX) and either mock-treated ( d ) or treated with damaging agents formaldehyde ( e ) or MMC ( f ) for different lengths of time at the indicated concentrations. At each time point, whole cell extracts were analysed by western blot to assess protein levels. Experiment has been done in triplicate. g Survival curves of HeLa FANCI −/− cells from clone 1 that were transfected with the different constructs of Flag-FANCI. Cell viability was monitored following cisplatin or olaparib treatments for 72 h and was assessed by counting remaining nuclei. Curves represent mean with SEM of three biological replicates. Western blots were used to monitor expression and shown here as an example. Alpha-tubulin was used as a loading control. Full blots are shown in Additional file

Article Snippet: After 18 h, cells were seeded on a glass coverslip for 8 h and then treated with 50 ng/ml MMC for 18 h and processed for immunofluorescence with anti-FANCD2 (Novus, ref NB100-182D1, 1:1000) antibody according to the protocol provided by Cell Signaling Technologies for Flag antibody (ref 8146).

Techniques: Variant Assay, Western Blot, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Cytidine deaminase protects pancreatic cancer cells from replicative stress and drives resistance to DNA-targeting drugs

doi: 10.1101/2021.10.23.465566

Figure Lengend Snippet: A. Immunofluorescence detection of FANCD2 foci (green), DAPI (blue). B . Quantification of the number of FANCD2 foci in early mitotic cells (prophase, metaphase) in at least 100 control cells, or cells overexpressing CDA (MIA PaCa-2) or depleted for CDA (CDA hairpins, Capan-1). ****: p<0.0001 (Wilcoxon-Mann-Whitney test). C. Fluorescence detection of micronuclei using DAPI staining, white arrows show micronuclei. D . quantification of the percentage of MIA PaCa-2 and BxPC-3 cells with at least one micronucleus. Results are representative of three independent transduction pools. *: p<0.05 (Wilcoxon-Mann-Whitney test). E. Immunofluorescence detection of 53BP1 bodies (green) and DAPI (blue). F . Quantification of the number of 53BP1 bodies in G1 cells in at least 1000 control cells and cells expressing CDA hairpins. Results are representative of three independent transduction pools. ***: p<0.001 (Wilcoxon-Mann-Whitney test).

Article Snippet: Primary antibodies were mouse anti-phospho-Histone (Ser139) (JBW301, Milipore 05-636, 1:1000), rabbit anti-phospho-53BP1 (S1778) (Cell Signaling #2675, 1:1000), rabbit anti-phospho-RPA2 (S4-S8) (Bethyl A300-245A, 1:500), rabbit anti-RPA70 (Cell Signaling #2267, 1:500), rabbit anti-FANCD2 (NOVUSBIO NB100-182, 1:1000) and rabbit anti-53BP1 (Bethyl A300-272A, 1:2500).

Techniques: Immunofluorescence, MANN-WHITNEY, Fluorescence, Staining, Transduction, Expressing