fancd2  (Cell Signaling Technology Inc)


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    ( 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 <t>FANCD2</t> 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.
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036970

    ( 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.
    Figure Legend 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.

    Techniques Used: Western Blot, Irradiation

    fancd2  (Cell Signaling Technology Inc)


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    ( 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 <t>FANCD2</t> 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.
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036970

    ( 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.
    Figure Legend 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.

    Techniques Used: Western Blot, Irradiation

    fancd2  (Cell Signaling Technology Inc)


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    (A) Formation of <t>FANCD2</t> foci in control vs. IRIS-depleted HME cells exposed to cisplatin or MMC. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (B) Defects in formation of FANCD2 foci in IRIS-knockout fibroblasts following cisplatin or MMC treatment. IRIS genotypes of the fibroblasts are indicated. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (C) IRIS depletion resulted in diminished FANCA foci in HME cells following ICL formation. Quantifications of percentage of FANCA foci-positive cells are shown on the bottom. (D) IRIS was required for RAD51 foci after MMC treatment, whereas it was dispensable for etoposide-induced RAD51 foci. Quantifications of percentage of RAD51 foci-positive cells are shown on the bottom. Representative images of immunofluorescence of cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ns, not significant; **p < 0.01; ***p < 0.001. Scale bar, 20 μm.
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The BRCA1 isoform, BRCA1-IRIS, operates independently of the full-length BRCA1 in the Fanconi anemia pathway"

    Article Title: The BRCA1 isoform, BRCA1-IRIS, operates independently of the full-length BRCA1 in the Fanconi anemia pathway

    Journal: bioRxiv

    doi: 10.1101/2022.11.02.514969

    (A) Formation of FANCD2 foci in control vs. IRIS-depleted HME cells exposed to cisplatin or MMC. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (B) Defects in formation of FANCD2 foci in IRIS-knockout fibroblasts following cisplatin or MMC treatment. IRIS genotypes of the fibroblasts are indicated. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (C) IRIS depletion resulted in diminished FANCA foci in HME cells following ICL formation. Quantifications of percentage of FANCA foci-positive cells are shown on the bottom. (D) IRIS was required for RAD51 foci after MMC treatment, whereas it was dispensable for etoposide-induced RAD51 foci. Quantifications of percentage of RAD51 foci-positive cells are shown on the bottom. Representative images of immunofluorescence of cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ns, not significant; **p < 0.01; ***p < 0.001. Scale bar, 20 μm.
    Figure Legend Snippet: (A) Formation of FANCD2 foci in control vs. IRIS-depleted HME cells exposed to cisplatin or MMC. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (B) Defects in formation of FANCD2 foci in IRIS-knockout fibroblasts following cisplatin or MMC treatment. IRIS genotypes of the fibroblasts are indicated. Quantifications of percentage of FANCD2 foci-positive cells are shown on the bottom. (C) IRIS depletion resulted in diminished FANCA foci in HME cells following ICL formation. Quantifications of percentage of FANCA foci-positive cells are shown on the bottom. (D) IRIS was required for RAD51 foci after MMC treatment, whereas it was dispensable for etoposide-induced RAD51 foci. Quantifications of percentage of RAD51 foci-positive cells are shown on the bottom. Representative images of immunofluorescence of cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ns, not significant; **p < 0.01; ***p < 0.001. Scale bar, 20 μm.

    Techniques Used: Knock-Out, Immunofluorescence, Immunostaining, Two Tailed Test

    (A) IRIS associated with FANCD2 and FANCA after cisplatin or MMC treatment. Nuclear extracts (NEs) of the indicated HME cells were prepared after 24-hour drug treatment and used for immunoprecipitation (IP) with FANCD2 antibody followed by immunoblotting. (B) The BRCA1 intron 11-encoded tail of IRIS was important for FANCD2 interaction. Whole cell extracts (WCEs) of transfected 293T cells were used for immunoprecipitation (IP) with Myc antibody, and the immune complexes were analyzed by immunoblotting. (C) wt IRIS, but not Tr IRIS, interacted with FANCA. WCEs of transfected 293T cells were used for IP with Myc antibody followed by immunoblotting. (D) Tr IRIS was unable to associate with FANCD2 and certain subunits of the FA core complex following exposure of HME cells to cisplatin or MMC. After 24-hour drug treatment, NEs from the indicated HME cells were used for IP with HA antibody followed by immunoblotting. (E) The Tower domain of FANCD2 and the C-terminal tail of IRIS were required for FANCD2-IRIS interaction. WCEs of transfected 293T cells were used for IP with HA antibody followed by immunoblotting. A schematic representation of full-length (FL) and Tower domain-deletion (ΔT, ΔTower) mutant FANCD2 proteins is shown. Numbers indicate positions of amino acid residues of the FL protein.
    Figure Legend Snippet: (A) IRIS associated with FANCD2 and FANCA after cisplatin or MMC treatment. Nuclear extracts (NEs) of the indicated HME cells were prepared after 24-hour drug treatment and used for immunoprecipitation (IP) with FANCD2 antibody followed by immunoblotting. (B) The BRCA1 intron 11-encoded tail of IRIS was important for FANCD2 interaction. Whole cell extracts (WCEs) of transfected 293T cells were used for immunoprecipitation (IP) with Myc antibody, and the immune complexes were analyzed by immunoblotting. (C) wt IRIS, but not Tr IRIS, interacted with FANCA. WCEs of transfected 293T cells were used for IP with Myc antibody followed by immunoblotting. (D) Tr IRIS was unable to associate with FANCD2 and certain subunits of the FA core complex following exposure of HME cells to cisplatin or MMC. After 24-hour drug treatment, NEs from the indicated HME cells were used for IP with HA antibody followed by immunoblotting. (E) The Tower domain of FANCD2 and the C-terminal tail of IRIS were required for FANCD2-IRIS interaction. WCEs of transfected 293T cells were used for IP with HA antibody followed by immunoblotting. A schematic representation of full-length (FL) and Tower domain-deletion (ΔT, ΔTower) mutant FANCD2 proteins is shown. Numbers indicate positions of amino acid residues of the FL protein.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Mutagenesis

    (A) FANCD2 colocalized with wt, but not Tr, IRIS in ICL-induced foci. Arrows point to some IRIS/FANCD2 colocalized foci. Quantifications of percentage of cells positive for IRIS/FANCD2-overlapping foci are shown on the bottom. (B) Colocalization of FANCA and wt IRIS in ICL-induced foci. Arrows point to some IRIS/FANCA colocalized foci. Quantifications of percentage of cells positive for IRIS/FANCA-overlapping foci are shown on the bottom. (C and D) Tr IRIS failed to rescue FANCD2 (C) or FANCA (D) foci in IRIS-depleted HME cells exposed to cisplatin or MMC. Quantifications of percentage of FANCD2 or FANCA foci-positive cells are shown on the bottom. Representative images of immunofluorescence of HME cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Zoomed-in areas containing a single nucleus each are shown next to their corresponding merged panels in (A and B). Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ***p < 0.001. Scale bar, 20 μm.
    Figure Legend Snippet: (A) FANCD2 colocalized with wt, but not Tr, IRIS in ICL-induced foci. Arrows point to some IRIS/FANCD2 colocalized foci. Quantifications of percentage of cells positive for IRIS/FANCD2-overlapping foci are shown on the bottom. (B) Colocalization of FANCA and wt IRIS in ICL-induced foci. Arrows point to some IRIS/FANCA colocalized foci. Quantifications of percentage of cells positive for IRIS/FANCA-overlapping foci are shown on the bottom. (C and D) Tr IRIS failed to rescue FANCD2 (C) or FANCA (D) foci in IRIS-depleted HME cells exposed to cisplatin or MMC. Quantifications of percentage of FANCD2 or FANCA foci-positive cells are shown on the bottom. Representative images of immunofluorescence of HME cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Zoomed-in areas containing a single nucleus each are shown next to their corresponding merged panels in (A and B). Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ***p < 0.001. Scale bar, 20 μm.

    Techniques Used: Immunofluorescence, Immunostaining, Two Tailed Test

    (A) IRIS depletion resulted in reductions in mono-ubiquitylation of FANCD2 in response to ICL damage. NEs of the indicated HME cells were prepared after 24-hour drug treatment and subjected to immunoblotting. (B) Expression of wt, but not Tr, IRIS restored FANCD2 mono-ubiquitylation in response to ICL damage. After 24-hour drug treatment, NEs from the indicated HME cells were used for Western blotting analysis. (C) Expression of wt, but not Tr, IRIS in IRIS-depleted HME cells rescued hyper toxicity induced by ICL damage. HME cells expressing the indicated hairpin and cDNA were exposed to cisplatin or mitomycin C for 24 hours and growth was assayed with CellTiter-Glo after a total of 5 days. Data shown are normalized mean ± SD (n = 6) of two independent experiments with triplicate wells in each experiment. P values were obtained using a two-way ANOVA test. ns, not significant; **p < 0.01.
    Figure Legend Snippet: (A) IRIS depletion resulted in reductions in mono-ubiquitylation of FANCD2 in response to ICL damage. NEs of the indicated HME cells were prepared after 24-hour drug treatment and subjected to immunoblotting. (B) Expression of wt, but not Tr, IRIS restored FANCD2 mono-ubiquitylation in response to ICL damage. After 24-hour drug treatment, NEs from the indicated HME cells were used for Western blotting analysis. (C) Expression of wt, but not Tr, IRIS in IRIS-depleted HME cells rescued hyper toxicity induced by ICL damage. HME cells expressing the indicated hairpin and cDNA were exposed to cisplatin or mitomycin C for 24 hours and growth was assayed with CellTiter-Glo after a total of 5 days. Data shown are normalized mean ± SD (n = 6) of two independent experiments with triplicate wells in each experiment. P values were obtained using a two-way ANOVA test. ns, not significant; **p < 0.01.

    Techniques Used: Western Blot, Expressing

    (A) IRIS, but not p220, was required for FANCA foci in HME cells after MMC treatment. Quantifications of percentage of FANCA foci-positive cells are shown on the bottom. (B) p220 was dispensable for the ICL-induced IRIS-FANCA-FANCD2 complex. NEs of the indicated HME cells were prepared after 24-hour drug treatment and used for IP with FANCD2 antibody followed by immunoblotting. (C) Loss of IRIS expression impaired p220 foci in HME cells in response to cisplatin or MMC, but not to etoposide or HU. Quantifications of percentage of cells positive for p220 foci are shown on the right. (D) Expression of wt, but not Tr, IRIS rescued ICL-inducible p220 foci in IRIS-depleted HME cells. Quantifications of percentage of p220 foci-positive cells are shown on the right. Representative images of immunofluorescence of HME cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ns, not significant; **p < 0.01. Scale bar, 20 μm.
    Figure Legend Snippet: (A) IRIS, but not p220, was required for FANCA foci in HME cells after MMC treatment. Quantifications of percentage of FANCA foci-positive cells are shown on the bottom. (B) p220 was dispensable for the ICL-induced IRIS-FANCA-FANCD2 complex. NEs of the indicated HME cells were prepared after 24-hour drug treatment and used for IP with FANCD2 antibody followed by immunoblotting. (C) Loss of IRIS expression impaired p220 foci in HME cells in response to cisplatin or MMC, but not to etoposide or HU. Quantifications of percentage of cells positive for p220 foci are shown on the right. (D) Expression of wt, but not Tr, IRIS rescued ICL-inducible p220 foci in IRIS-depleted HME cells. Quantifications of percentage of p220 foci-positive cells are shown on the right. Representative images of immunofluorescence of HME cells treated with the indicated drug for 24 hours before immunostaining using the indicated antibodies are shown. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. ns, not significant; **p < 0.01. Scale bar, 20 μm.

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Immunostaining, Two Tailed Test

    (A) Cooperation between IRIS and FANCA in cell survival after cisplatin or mitomycin C treatment. The IRIS and/or FANCA genotypes of the engineered fibroblasts are indicated. Fibroblasts were exposed to the indicated drug for 24 hours and growth was assayed with CellTiter-Glo after a total of 5 days. Data shown are normalized mean ± SD (n = 6) of two independent experiments with triplicate wells in each experiment. P values were obtained using a two-way ANOVA test. (B) Abundance of relevant proteins in the engineered fibroblasts analyzed by immunoblotting. (C) Analysis of ICL-induced FANCD2 foci in the engineered fibroblasts. Representative images of immunofluorescence of cells treated with the indicated drug for 24 hours before anti-γH2A.X and anti-FANCD2 immunostaining are shown. Quantifications of percentage of FANCD2 foci-positive cells are shown on the right. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. Scale bar, 20 μm. ns, not significant; **p < 0.01.
    Figure Legend Snippet: (A) Cooperation between IRIS and FANCA in cell survival after cisplatin or mitomycin C treatment. The IRIS and/or FANCA genotypes of the engineered fibroblasts are indicated. Fibroblasts were exposed to the indicated drug for 24 hours and growth was assayed with CellTiter-Glo after a total of 5 days. Data shown are normalized mean ± SD (n = 6) of two independent experiments with triplicate wells in each experiment. P values were obtained using a two-way ANOVA test. (B) Abundance of relevant proteins in the engineered fibroblasts analyzed by immunoblotting. (C) Analysis of ICL-induced FANCD2 foci in the engineered fibroblasts. Representative images of immunofluorescence of cells treated with the indicated drug for 24 hours before anti-γH2A.X and anti-FANCD2 immunostaining are shown. Quantifications of percentage of FANCD2 foci-positive cells are shown on the right. Bars represent mean ± SD (n = 3) of three independent experiments. P values were obtained using a two-tailed Student’s t test. Scale bar, 20 μm. ns, not significant; **p < 0.01.

    Techniques Used: Western Blot, Immunofluorescence, Immunostaining, Two Tailed Test

    fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fancd2
    (A) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- <t>FANCD2</t> KO cells were treated with cisplatin for 24 h, mitomycin C for 24 h or olaparib for 7 days. Colony formation was measured after 7 days by Coomassie Brilliant Blue staining. Graphs represent mean and standard error of the mean (SEM) of three independent biological replicates. (B) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with 1 μM cisplatin for 24 h, and cells were fixed at 0 h, 8 h and 24 h after washout of cisplatin. Cells were stained for γH2AX foci formation, and at least 100 cells per condition were scored. Statistics were performed using two-tailed Mann-Whitney tests. (C) Cell cycle profiles of HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were generated by flow cytometry analysis of EdU and PI signal. Bars represent means of three replicates. (D) Copy number alterations (CNAs) were analysed using single-cell sequencing of 28 HAP1, 19 HAP1- C1orf112 KO and 28 HAP1- FIGNL1 KO libraries. Cisplatin treatment was performed with 1 μM cisplatin for 96 h. CNA analysis in cisplatin-treated cells was performed using 24 HAP1, 31 HAP1- C1orf112 KO and 27 HAP1- FIGNL1 KO libraries. Statistics were performed using two-tailed Mann-Whitney tests. (E) PICH-positive anaphase bridges were analysed in unchallenged HAP1-sgLUC, HAP1- C1orf112 KO and HAP1- FIGNL1 KO cells. At least 60 anaphases were scored per condition.
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The FIGNL1-interacting protein C1orf112 is synthetic lethal with PICH and mediates RAD51 retention on chromatin"

    Article Title: The FIGNL1-interacting protein C1orf112 is synthetic lethal with PICH and mediates RAD51 retention on chromatin

    Journal: bioRxiv

    doi: 10.1101/2022.10.07.511242

    (A) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with cisplatin for 24 h, mitomycin C for 24 h or olaparib for 7 days. Colony formation was measured after 7 days by Coomassie Brilliant Blue staining. Graphs represent mean and standard error of the mean (SEM) of three independent biological replicates. (B) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with 1 μM cisplatin for 24 h, and cells were fixed at 0 h, 8 h and 24 h after washout of cisplatin. Cells were stained for γH2AX foci formation, and at least 100 cells per condition were scored. Statistics were performed using two-tailed Mann-Whitney tests. (C) Cell cycle profiles of HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were generated by flow cytometry analysis of EdU and PI signal. Bars represent means of three replicates. (D) Copy number alterations (CNAs) were analysed using single-cell sequencing of 28 HAP1, 19 HAP1- C1orf112 KO and 28 HAP1- FIGNL1 KO libraries. Cisplatin treatment was performed with 1 μM cisplatin for 96 h. CNA analysis in cisplatin-treated cells was performed using 24 HAP1, 31 HAP1- C1orf112 KO and 27 HAP1- FIGNL1 KO libraries. Statistics were performed using two-tailed Mann-Whitney tests. (E) PICH-positive anaphase bridges were analysed in unchallenged HAP1-sgLUC, HAP1- C1orf112 KO and HAP1- FIGNL1 KO cells. At least 60 anaphases were scored per condition.
    Figure Legend Snippet: (A) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with cisplatin for 24 h, mitomycin C for 24 h or olaparib for 7 days. Colony formation was measured after 7 days by Coomassie Brilliant Blue staining. Graphs represent mean and standard error of the mean (SEM) of three independent biological replicates. (B) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with 1 μM cisplatin for 24 h, and cells were fixed at 0 h, 8 h and 24 h after washout of cisplatin. Cells were stained for γH2AX foci formation, and at least 100 cells per condition were scored. Statistics were performed using two-tailed Mann-Whitney tests. (C) Cell cycle profiles of HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were generated by flow cytometry analysis of EdU and PI signal. Bars represent means of three replicates. (D) Copy number alterations (CNAs) were analysed using single-cell sequencing of 28 HAP1, 19 HAP1- C1orf112 KO and 28 HAP1- FIGNL1 KO libraries. Cisplatin treatment was performed with 1 μM cisplatin for 96 h. CNA analysis in cisplatin-treated cells was performed using 24 HAP1, 31 HAP1- C1orf112 KO and 27 HAP1- FIGNL1 KO libraries. Statistics were performed using two-tailed Mann-Whitney tests. (E) PICH-positive anaphase bridges were analysed in unchallenged HAP1-sgLUC, HAP1- C1orf112 KO and HAP1- FIGNL1 KO cells. At least 60 anaphases were scored per condition.

    Techniques Used: Staining, Two Tailed Test, MANN-WHITNEY, Generated, Flow Cytometry, Sequencing

    (A) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with 1 μM cisplatin for 24 h, and cells were at fixed 0 h, 8 h and 24 h after washout of cisplatin. Cells were stained for RAD51, and at least 100 cells per condition were scored for foci formation. Statistics were performed using two-tailed Mann-Whitney tests. (B) HAP1-sgLUC, HAP1- C1orf112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and whole-cell lysates, cytosolic, nuclear and chromatin protein fractions were subsequently isolated. Expression of RAD51, HSP90 (cytosolic marker), LaminB1 (nuclear marker), and Histone H3 (chromatin marker) were analysed by western blot. (C) HeLa DR-GFP cells were transfected with siRNAs targeting BRCA2, C1orf112 or FIGNL1. 48 h after transection, expression of BRCA2, C1orf112 and FIGNL1 was analysed by western blot. (D) HeLa DR-GFP cells were sequentially transfected with the indicated siRNAs and I-SceI plasmid. 48 h after I-SceI transfection, GFP-positivity was analysed by flow cytometry. Data points represent three independent biological replicates and standard error of the mean (SEM). Statistics were performed by Mann-Whitney tests. (E) HAP1-sgLUC, HAP1- C1ORF112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were pulse-labelled with IdU, followed by and CldU in the presence of 4 mM HU. Lengths of at least 200 DNA fibres were measured per condition. Statistics were performed by two-tailed Mann-Whitney tests. (F) HAP1 cells were labeled with EdU for 10 minutes. For chase conditions, EdU labeling was followed with thymidine treatment for 1 h. Fork stalling was induced by treatment with 4 mM HU for 2 h. Proteins at nascent DNA were cross-linked using formaldehyde, EdU was coupled to biotin-conjugated azide, and protein capture was performed using streptavidin beads. C1orf112, FIGNL1, PCNA, Histone H3 and γH2AX levels in input and iPOND fractions were analysed by western blot. (G) HAP1-sgLUC, HAP1- C1ORF112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and subsequently FANCD2 foci were scored in at least 30 mitotic cells were analysed in three replicates. Statistics were performed by one-way Anova. (H) HAP1-sgLUC, HAP1- C1ORF112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and subsequently 53BP1 bodies were analysed in at least 250 cells per condition per replicate. Statistics were performed using one-way Anova with Sidak’s post hoc test.
    Figure Legend Snippet: (A) HAP1-sgLUC, HAP1- C1orf112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were treated with 1 μM cisplatin for 24 h, and cells were at fixed 0 h, 8 h and 24 h after washout of cisplatin. Cells were stained for RAD51, and at least 100 cells per condition were scored for foci formation. Statistics were performed using two-tailed Mann-Whitney tests. (B) HAP1-sgLUC, HAP1- C1orf112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and whole-cell lysates, cytosolic, nuclear and chromatin protein fractions were subsequently isolated. Expression of RAD51, HSP90 (cytosolic marker), LaminB1 (nuclear marker), and Histone H3 (chromatin marker) were analysed by western blot. (C) HeLa DR-GFP cells were transfected with siRNAs targeting BRCA2, C1orf112 or FIGNL1. 48 h after transection, expression of BRCA2, C1orf112 and FIGNL1 was analysed by western blot. (D) HeLa DR-GFP cells were sequentially transfected with the indicated siRNAs and I-SceI plasmid. 48 h after I-SceI transfection, GFP-positivity was analysed by flow cytometry. Data points represent three independent biological replicates and standard error of the mean (SEM). Statistics were performed by Mann-Whitney tests. (E) HAP1-sgLUC, HAP1- C1ORF112 KO , HAP1- FIGNL1 KO and HAP1- FANCD2 KO cells were pulse-labelled with IdU, followed by and CldU in the presence of 4 mM HU. Lengths of at least 200 DNA fibres were measured per condition. Statistics were performed by two-tailed Mann-Whitney tests. (F) HAP1 cells were labeled with EdU for 10 minutes. For chase conditions, EdU labeling was followed with thymidine treatment for 1 h. Fork stalling was induced by treatment with 4 mM HU for 2 h. Proteins at nascent DNA were cross-linked using formaldehyde, EdU was coupled to biotin-conjugated azide, and protein capture was performed using streptavidin beads. C1orf112, FIGNL1, PCNA, Histone H3 and γH2AX levels in input and iPOND fractions were analysed by western blot. (G) HAP1-sgLUC, HAP1- C1ORF112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and subsequently FANCD2 foci were scored in at least 30 mitotic cells were analysed in three replicates. Statistics were performed by one-way Anova. (H) HAP1-sgLUC, HAP1- C1ORF112 KO and HAP1- FIGNL1 KO cells were treated with 1 μM cisplatin for 24 h, and subsequently 53BP1 bodies were analysed in at least 250 cells per condition per replicate. Statistics were performed using one-way Anova with Sidak’s post hoc test.

    Techniques Used: Staining, Two Tailed Test, MANN-WHITNEY, Isolation, Expressing, Marker, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Labeling

    fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fancd2
    A Cell proliferation assay of wild type , <t>Fancd2</t> −/− exposed to the indicated dose of 5dC, 5mdC, 5hmdC, 5fdC and 5cadC for 3 days ( n = 4, mean ± s.d.). Cell proliferation assay of a lymphoblast FANCA-deficient patient-derived HSC72 , NV012 cell lines and FANCD2 − KO eHAP CRISPR clones exposed to the indicated doses of 5hmdC for 3 days ( n = 4, mean ± s.d.). B Top left , representative PAR (red) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Top right , plot depicting PAR mean intensity signal per nucleus ( n = 3, Mann–Whitney test; central line represents median value). Bottom left , representative γ-H2AX (green) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Bottom right , plot depicting γ-H2AX foci per nucleus ( n = 3, Student’s t test; central line represents mean ± s.d.). C Left , representative images of chromosome aberration test (red arrowhead) from wild type and Fancd2 −/− cells following 5hmdC treatment (10 μM) for 40 h. Right , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.e.m.). D Western blot of wild type and Fancd2 −/− MEFs extracts to detect γ-H2AX, ser345-CHK1, total CHK1, and PCNA (loading control) after exposure to 5dC, 5mdC, 5hmdC, 5fdC or 5cadC (10 μM) for 16 h. E Bar plot depicting frequency of G1, S or G2 populations of wild type and Fancd2 −/− cells exposed to 5dC, 5mdC and 5hmdC (100 μM) for 30 min and subsequently analyzed after 48 h in culture ( n = 3, Student’s t test; bar represents mean ± s.d.). F Left , plot depicting γ-H2AX foci per nucleus of wild type and Fancd2 −/− cells exposed to AZD7762 (2.5 nM), 5hmdC (10 μM) or combination of both for 16 h ( n = 3, Mann–Whitney test; central line represents mean value). Right , as in Left but AZD7762 was substituted by UCN-01 (2.5 nM). G Bar plot of breakdown of the different types of chromosomal aberrations from wild type and Fancd2 −/− cells treated with 5hmdC (10 μM), AZD7762 (2.5 nM), UCN-01 (2.5 nM) or combinations for 48 h. AZD7762 or UCN-01 were added 24 h before harvesting the cells ( n = 100 of each of 2 biological replicates, Student’s t test; bar represents mean ± s.d).
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    1) Product Images from "FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine"

    Article Title: FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04952-0

    A Cell proliferation assay of wild type , Fancd2 −/− exposed to the indicated dose of 5dC, 5mdC, 5hmdC, 5fdC and 5cadC for 3 days ( n = 4, mean ± s.d.). Cell proliferation assay of a lymphoblast FANCA-deficient patient-derived HSC72 , NV012 cell lines and FANCD2 − KO eHAP CRISPR clones exposed to the indicated doses of 5hmdC for 3 days ( n = 4, mean ± s.d.). B Top left , representative PAR (red) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Top right , plot depicting PAR mean intensity signal per nucleus ( n = 3, Mann–Whitney test; central line represents median value). Bottom left , representative γ-H2AX (green) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Bottom right , plot depicting γ-H2AX foci per nucleus ( n = 3, Student’s t test; central line represents mean ± s.d.). C Left , representative images of chromosome aberration test (red arrowhead) from wild type and Fancd2 −/− cells following 5hmdC treatment (10 μM) for 40 h. Right , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.e.m.). D Western blot of wild type and Fancd2 −/− MEFs extracts to detect γ-H2AX, ser345-CHK1, total CHK1, and PCNA (loading control) after exposure to 5dC, 5mdC, 5hmdC, 5fdC or 5cadC (10 μM) for 16 h. E Bar plot depicting frequency of G1, S or G2 populations of wild type and Fancd2 −/− cells exposed to 5dC, 5mdC and 5hmdC (100 μM) for 30 min and subsequently analyzed after 48 h in culture ( n = 3, Student’s t test; bar represents mean ± s.d.). F Left , plot depicting γ-H2AX foci per nucleus of wild type and Fancd2 −/− cells exposed to AZD7762 (2.5 nM), 5hmdC (10 μM) or combination of both for 16 h ( n = 3, Mann–Whitney test; central line represents mean value). Right , as in Left but AZD7762 was substituted by UCN-01 (2.5 nM). G Bar plot of breakdown of the different types of chromosomal aberrations from wild type and Fancd2 −/− cells treated with 5hmdC (10 μM), AZD7762 (2.5 nM), UCN-01 (2.5 nM) or combinations for 48 h. AZD7762 or UCN-01 were added 24 h before harvesting the cells ( n = 100 of each of 2 biological replicates, Student’s t test; bar represents mean ± s.d).
    Figure Legend Snippet: A Cell proliferation assay of wild type , Fancd2 −/− exposed to the indicated dose of 5dC, 5mdC, 5hmdC, 5fdC and 5cadC for 3 days ( n = 4, mean ± s.d.). Cell proliferation assay of a lymphoblast FANCA-deficient patient-derived HSC72 , NV012 cell lines and FANCD2 − KO eHAP CRISPR clones exposed to the indicated doses of 5hmdC for 3 days ( n = 4, mean ± s.d.). B Top left , representative PAR (red) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Top right , plot depicting PAR mean intensity signal per nucleus ( n = 3, Mann–Whitney test; central line represents median value). Bottom left , representative γ-H2AX (green) immunofluorescence images of wild type and Fancd2 −/− cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Bottom right , plot depicting γ-H2AX foci per nucleus ( n = 3, Student’s t test; central line represents mean ± s.d.). C Left , representative images of chromosome aberration test (red arrowhead) from wild type and Fancd2 −/− cells following 5hmdC treatment (10 μM) for 40 h. Right , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.e.m.). D Western blot of wild type and Fancd2 −/− MEFs extracts to detect γ-H2AX, ser345-CHK1, total CHK1, and PCNA (loading control) after exposure to 5dC, 5mdC, 5hmdC, 5fdC or 5cadC (10 μM) for 16 h. E Bar plot depicting frequency of G1, S or G2 populations of wild type and Fancd2 −/− cells exposed to 5dC, 5mdC and 5hmdC (100 μM) for 30 min and subsequently analyzed after 48 h in culture ( n = 3, Student’s t test; bar represents mean ± s.d.). F Left , plot depicting γ-H2AX foci per nucleus of wild type and Fancd2 −/− cells exposed to AZD7762 (2.5 nM), 5hmdC (10 μM) or combination of both for 16 h ( n = 3, Mann–Whitney test; central line represents mean value). Right , as in Left but AZD7762 was substituted by UCN-01 (2.5 nM). G Bar plot of breakdown of the different types of chromosomal aberrations from wild type and Fancd2 −/− cells treated with 5hmdC (10 μM), AZD7762 (2.5 nM), UCN-01 (2.5 nM) or combinations for 48 h. AZD7762 or UCN-01 were added 24 h before harvesting the cells ( n = 100 of each of 2 biological replicates, Student’s t test; bar represents mean ± s.d).

    Techniques Used: Proliferation Assay, Derivative Assay, CRISPR, Clone Assay, Immunofluorescence, MANN-WHITNEY, Western Blot

    A HPLC-MS/MS quantitation of endogenous and exogenous 5hmdC levels in genomic DNA samples of wild type and Fancd2 −/− cells exposed to D3-labeled 5hmdC (0, 10 and 20 μM) for 16 h. Wild type cells showed a 0.49 vs 4.8 exogenous vs endogenous 5hmdC per 10 4 dG upon 10 μM 5hmdC-D3 exposure; 1.2 5hmdC-D3 vs 4.5 5hmdC per 10 4 dG for 20 μM exposure). Fancd2 −/− cells showed a 3.2 5hmdC-D3 vs 5.8 5hmdC per 10 4 dG upon 10 μM 5hmdC-D3 exposure; 2.82 5hmdC-D3 vs 4.91 5hmdC per 10 4 dG upon 20 μM 5hmdC-D3 exposure. Plots represent mean values from 3 biological replicates. B Left , representative immunofluorescence of wild type and Fancd2 −/− MEFs showing PAR nuclear staining and EdU positive (EdU+) cells after exposure to 5hmdC (10 μM) for 16 h ( n = 3). Right , bar plot showing percentage of PAR+ cells in EdU negative (EdU−) or EdU+ cell populations ( n = 250 of each of 3 independent biological replicates, Student’s t test; bars represents mean ± s.d.). C Western blot of iPOND-captured and input protein extracts from wild type and Fancd2 −/− cells treated with 5hmdC (160 μM) or HU (1 mM) for 3 h to detect γ-H2AX, PCNA and RAD51. D Top left , scheme of BrdU incorporation assay. Bottom left , representative immunofluorescence images of wild type and Fancd2 −/− MEFs showing BrdU + ( Red ) or BrdU- cells and nuclear DNA (DAPI, blue ). Right , percentage of BrdU+ or BrdU− cells after HU (1 mM) or 5hmdC (40 μM) for 16 h ( n = 200 of each of 3 biological replicates, Student’s t test; Bar represents mean ± s.d.). E Top , plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2 −/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h ( n = 3, Mann–Whitney test; central line represents median value). Bottom , plot depicting γ-H2AX foci per nucleus of wild type and Fancd2 −/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h ( n = 3, Student’s t test; central line represents mean ± s.d.). F Top left , scheme of the DNA fiber assay. Bottom left , representative images of DNA fibers from wild type and Fancd2 −/− MEFs after 10, 40, 80 or 160 μM 5hmdC exposure. Right , plot representing fork speed (Kbp min −1 ) of wild type and Fancd2 −/− MEFs exposed to 10, 40, 80 or 160 μM 5hmdC ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value).
    Figure Legend Snippet: A HPLC-MS/MS quantitation of endogenous and exogenous 5hmdC levels in genomic DNA samples of wild type and Fancd2 −/− cells exposed to D3-labeled 5hmdC (0, 10 and 20 μM) for 16 h. Wild type cells showed a 0.49 vs 4.8 exogenous vs endogenous 5hmdC per 10 4 dG upon 10 μM 5hmdC-D3 exposure; 1.2 5hmdC-D3 vs 4.5 5hmdC per 10 4 dG for 20 μM exposure). Fancd2 −/− cells showed a 3.2 5hmdC-D3 vs 5.8 5hmdC per 10 4 dG upon 10 μM 5hmdC-D3 exposure; 2.82 5hmdC-D3 vs 4.91 5hmdC per 10 4 dG upon 20 μM 5hmdC-D3 exposure. Plots represent mean values from 3 biological replicates. B Left , representative immunofluorescence of wild type and Fancd2 −/− MEFs showing PAR nuclear staining and EdU positive (EdU+) cells after exposure to 5hmdC (10 μM) for 16 h ( n = 3). Right , bar plot showing percentage of PAR+ cells in EdU negative (EdU−) or EdU+ cell populations ( n = 250 of each of 3 independent biological replicates, Student’s t test; bars represents mean ± s.d.). C Western blot of iPOND-captured and input protein extracts from wild type and Fancd2 −/− cells treated with 5hmdC (160 μM) or HU (1 mM) for 3 h to detect γ-H2AX, PCNA and RAD51. D Top left , scheme of BrdU incorporation assay. Bottom left , representative immunofluorescence images of wild type and Fancd2 −/− MEFs showing BrdU + ( Red ) or BrdU- cells and nuclear DNA (DAPI, blue ). Right , percentage of BrdU+ or BrdU− cells after HU (1 mM) or 5hmdC (40 μM) for 16 h ( n = 200 of each of 3 biological replicates, Student’s t test; Bar represents mean ± s.d.). E Top , plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2 −/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h ( n = 3, Mann–Whitney test; central line represents median value). Bottom , plot depicting γ-H2AX foci per nucleus of wild type and Fancd2 −/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h ( n = 3, Student’s t test; central line represents mean ± s.d.). F Top left , scheme of the DNA fiber assay. Bottom left , representative images of DNA fibers from wild type and Fancd2 −/− MEFs after 10, 40, 80 or 160 μM 5hmdC exposure. Right , plot representing fork speed (Kbp min −1 ) of wild type and Fancd2 −/− MEFs exposed to 10, 40, 80 or 160 μM 5hmdC ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value).

    Techniques Used: Tandem Mass Spectroscopy, Quantitation Assay, Labeling, Immunofluorescence, Staining, Western Blot, BrdU Incorporation Assay, MANN-WHITNEY

    A Dot plot showing quantitation of nuclear γ-H2AX foci in wild type and Fancd2 −/− MEFs upon treatment with 5hmdC (2 μM), olaparib (7.5 nM) or 5hmdC+olaparib for 16 h ( n = 100 of each of 3 biological replicates, Student’s t test; central line represents mean ± s.d.). B Left , representative images of chromosome metaphases from wild type and Fancd2 −/− cells treated with 5hmdC (2 μM), olaparib (7.5 nM) or 5hmdC+olaparib. Right , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.d.). C Dot plot representing immunostaining of PARP1 during the chromatin retention assay from wild type and Fancd2 −/− cells exposed to 5hmdC (10, 20, 40 μM) for 3 h ( left ) or in combination with olaparib (0.1 μM + 10 μM 5hmdC) ( right ). Olaparib was added 30 min before 5hmdC ( n = 3, Mann–Whitney test; central line represents median value). D Cell proliferation assay of Parp1 +/+ and Parp1 −/− MEFs exposed to the indicated doses of 5hmdC for 4 days ( n = 4, mean ± s.d.). E Top left , scheme of the DNA fiber assay of wild type and Fancd2 −/− cells in the presence of 5hmdC in combination with olaparib. Bottom left , representative images of DNA fibers from wild type and Fancd2 −/− MEFs after 5hmdC (40 μM) exposure alone or in combination with olaparib (0.5 μM). Right , plot representing fork speed (Kbp min −1 ) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). F Dot plot representing fork speed (Kbp min −1 ) of Parp1 +/+ and Parp1 −/− in the presence of 5hmdC (40 μM)( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). G Left , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdC alone or combined with a fixed dose of olaparib (15 nM), for 3 days ( n = 4, mean ± s.d.), or right , to the indicated doses of olaparib alone or combined with a fixed dose of 5hmdC (1.25 μM) for 3 days ( n = 4, mean ± s.d.).
    Figure Legend Snippet: A Dot plot showing quantitation of nuclear γ-H2AX foci in wild type and Fancd2 −/− MEFs upon treatment with 5hmdC (2 μM), olaparib (7.5 nM) or 5hmdC+olaparib for 16 h ( n = 100 of each of 3 biological replicates, Student’s t test; central line represents mean ± s.d.). B Left , representative images of chromosome metaphases from wild type and Fancd2 −/− cells treated with 5hmdC (2 μM), olaparib (7.5 nM) or 5hmdC+olaparib. Right , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.d.). C Dot plot representing immunostaining of PARP1 during the chromatin retention assay from wild type and Fancd2 −/− cells exposed to 5hmdC (10, 20, 40 μM) for 3 h ( left ) or in combination with olaparib (0.1 μM + 10 μM 5hmdC) ( right ). Olaparib was added 30 min before 5hmdC ( n = 3, Mann–Whitney test; central line represents median value). D Cell proliferation assay of Parp1 +/+ and Parp1 −/− MEFs exposed to the indicated doses of 5hmdC for 4 days ( n = 4, mean ± s.d.). E Top left , scheme of the DNA fiber assay of wild type and Fancd2 −/− cells in the presence of 5hmdC in combination with olaparib. Bottom left , representative images of DNA fibers from wild type and Fancd2 −/− MEFs after 5hmdC (40 μM) exposure alone or in combination with olaparib (0.5 μM). Right , plot representing fork speed (Kbp min −1 ) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). F Dot plot representing fork speed (Kbp min −1 ) of Parp1 +/+ and Parp1 −/− in the presence of 5hmdC (40 μM)( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). G Left , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdC alone or combined with a fixed dose of olaparib (15 nM), for 3 days ( n = 4, mean ± s.d.), or right , to the indicated doses of olaparib alone or combined with a fixed dose of 5hmdC (1.25 μM) for 3 days ( n = 4, mean ± s.d.).

    Techniques Used: Quantitation Assay, Immunostaining, MANN-WHITNEY, Proliferation Assay

    A Plot representing the quantitation by HPLC-MS/MS of exogenous 5hmdU-D3 level in genomic DNA samples from wild type and Fancd2 −/− cells exposed to isotopically labeled 5hmdC-D3 (0,10 and 20 μM) for 16 h. B Top , plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2 −/− cells exposed to 5hmdU (2.5 μM) for 16 h ( n = 3, Mann–Whitney test; central line represents median value). Bottom , plot depicting γ-H2AX foci obtained from immunofluorescence images from wild type or Fancd2 −/− cells exposed to 5hmdU (2.5 μM) for 16 h ( n = 3, Student’s t test; central line represents mean ± s.d.). C Top , representative images of chromosome aberrations (red arrowhead) test from wild type and Fancd2 −/− cells following 5hmdU treatment (1.25 and 2.5 μM) for 40 h. Bottom , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.d.). D Cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdU for 3 days ( n = 4, mean ± s.d.). E Cell proliferation assays of MDA-MB-231 (BRCA1 proficient), MDA-MB-436 (BRCA1 deficient), BRCA2 +/+ and BRCA2 −/− DLD-1 cell exposed to the indicated doses of 5hmdC or 5hmdU for 7 days ( n = 4, mean ± s.d).
    Figure Legend Snippet: A Plot representing the quantitation by HPLC-MS/MS of exogenous 5hmdU-D3 level in genomic DNA samples from wild type and Fancd2 −/− cells exposed to isotopically labeled 5hmdC-D3 (0,10 and 20 μM) for 16 h. B Top , plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2 −/− cells exposed to 5hmdU (2.5 μM) for 16 h ( n = 3, Mann–Whitney test; central line represents median value). Bottom , plot depicting γ-H2AX foci obtained from immunofluorescence images from wild type or Fancd2 −/− cells exposed to 5hmdU (2.5 μM) for 16 h ( n = 3, Student’s t test; central line represents mean ± s.d.). C Top , representative images of chromosome aberrations (red arrowhead) test from wild type and Fancd2 −/− cells following 5hmdU treatment (1.25 and 2.5 μM) for 40 h. Bottom , bar plot of breakdown of the different types of chromosomal aberrations ( n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.d.). D Cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdU for 3 days ( n = 4, mean ± s.d.). E Cell proliferation assays of MDA-MB-231 (BRCA1 proficient), MDA-MB-436 (BRCA1 deficient), BRCA2 +/+ and BRCA2 −/− DLD-1 cell exposed to the indicated doses of 5hmdC or 5hmdU for 7 days ( n = 4, mean ± s.d).

    Techniques Used: Quantitation Assay, Tandem Mass Spectroscopy, Labeling, MANN-WHITNEY, Immunofluorescence, Proliferation Assay

    A Dot plot representing fork speed (Kbp min −1 ) of wild type and Fancd2 −/− cells exposed to 5hmdU (40 μM) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). B Left , dot plot representing the PARP1 chromatin retention assay from wild type and Fancd2 −/− cells upon exposure to 5hmdU (0, 10, 20 and 40 μM) for 3 h. Right , PARP1 chromatin retention assay of wild type and Fancd2 −/− cells exposed to 5hmdU (10 μm), olaparib (0.1 μM) or combination of 5hmdU + olaparib for 3 h. C Dot plot representing replication fork speed of wild type and Fancd2 −/− cells exposed to 5hmdU (40 μM), olaparib (0.5 μM) or a combination of 5hmdU + Olaparib. Olaparib was added 60 min before adding CldU ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). D Dot plot representing replication fork speed of Parp1 +/+ and Parp1 −/− cells exposed to 5hmdU (40 μM) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). E Top , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdU alone or combined with a fixed dose of olaparib (15 nM) for 3 days ( n = 4, mean ± s.d.). Bottom , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of olaparib alone or combined with a fixed dose of 5hmdU (1.25 μM), for 3 days ( n = 4, mean ± s.d.). F Cell proliferation assay of Parp1 +/+ and Parp1 −/− MEFs exposed to the indicated doses of olaparib combined with a fixed dose (1.25 μM) of either 5hmdC ( left ) or 5hmdU ( right ) for 3 days ( n = 4, mean ± s.d.). G Model depicting the instability of replication fork in the absence of FANCD2, caused by the recruitment of PARP1 to sites of 5hmdC or 5hmdU removal by the excision repair machinery.
    Figure Legend Snippet: A Dot plot representing fork speed (Kbp min −1 ) of wild type and Fancd2 −/− cells exposed to 5hmdU (40 μM) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). B Left , dot plot representing the PARP1 chromatin retention assay from wild type and Fancd2 −/− cells upon exposure to 5hmdU (0, 10, 20 and 40 μM) for 3 h. Right , PARP1 chromatin retention assay of wild type and Fancd2 −/− cells exposed to 5hmdU (10 μm), olaparib (0.1 μM) or combination of 5hmdU + olaparib for 3 h. C Dot plot representing replication fork speed of wild type and Fancd2 −/− cells exposed to 5hmdU (40 μM), olaparib (0.5 μM) or a combination of 5hmdU + Olaparib. Olaparib was added 60 min before adding CldU ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). D Dot plot representing replication fork speed of Parp1 +/+ and Parp1 −/− cells exposed to 5hmdU (40 μM) ( n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value). E Top , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of 5hmdU alone or combined with a fixed dose of olaparib (15 nM) for 3 days ( n = 4, mean ± s.d.). Bottom , cell proliferation assay of wild type and Fancd2 −/− MEFs exposed to the indicated doses of olaparib alone or combined with a fixed dose of 5hmdU (1.25 μM), for 3 days ( n = 4, mean ± s.d.). F Cell proliferation assay of Parp1 +/+ and Parp1 −/− MEFs exposed to the indicated doses of olaparib combined with a fixed dose (1.25 μM) of either 5hmdC ( left ) or 5hmdU ( right ) for 3 days ( n = 4, mean ± s.d.). G Model depicting the instability of replication fork in the absence of FANCD2, caused by the recruitment of PARP1 to sites of 5hmdC or 5hmdU removal by the excision repair machinery.

    Techniques Used: MANN-WHITNEY, Proliferation Assay

    anti fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti fancd2
    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 <t>FANCD2</t> 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
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    1) Product Images from "A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene"

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

    Journal: Genome Medicine

    doi: 10.1186/s13073-021-00998-5

    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
    Figure Legend 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

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

    anti fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti fancd2
    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 <t>FANCD2</t> 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
    Anti Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene"

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

    Journal: Genome Medicine

    doi: 10.1186/s13073-021-00998-5

    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
    Figure Legend 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

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

    rabbit anti fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti fancd2
    A. Immunofluorescence detection of <t>FANCD2</t> 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).
    Rabbit Anti Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cytidine deaminase protects pancreatic cancer cells from replicative stress and drives resistance to DNA-targeting drugs"

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

    Journal: bioRxiv

    doi: 10.1101/2021.10.23.465566

    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).
    Figure Legend 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).

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

    fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fancd2
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc fancd2
    Fancd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fancd2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fancd2
    Recruitment of various DDR proteins to lacO arrays after transient expression of HA-LacI in U2OS 2–6-3 cells. (A) U2OS 2–6-3 cells were transfected with HA-LacI expression vector for 24 h using Lipofectamine 2000 reagent (Invitrogen) and then double immunostained with the indicated antibodies, followed by DAPI staining. Representative images and colocalization frequencies (%) of DDR-protein foci with LacI foci are shown. Values were calculated from the sum scores of at least two independent experiments. Scale bars, 10 µm. (B) U2OS 2–6-3 cells were transfected with the empty or the HA-LacI expression vectors for 24 h and then subjected to ChIP–quantitative PCR analysis with the indicated antibodies. Quantitative PCR was performed with primer pairs to detect lacO sequences. Results are shown as the percentage of input DNA. Means ± SD are shown ( n = 4). *, P < 0.05; **, P < 0.01; n.s., not significant (two-tailed Student’s t test). (C and D) U2OS 2–6-3 cells transfected as in (B) were treated with 10 µM VE-821 (ATR/ATM inhibitor), 10 µM KU-55933 (ATM inhibitor), or a control vehicle (DMSO) for 6 h and then subjected to immunoblotting. Data are representative of two or more independent experiments. In this experimental condition, VE-821 selectively inhibited ATR-mediated phosphorylation of Chk1 (Ser345), but not autophosphorylation of ATM (Ser1981). CBB, Coomassie Brilliant Blue; <t>FANCD2-Ub,</t> monoubiquitinated FANCD2.
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    1) Product Images from "SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions"

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202003148

    Recruitment of various DDR proteins to lacO arrays after transient expression of HA-LacI in U2OS 2–6-3 cells. (A) U2OS 2–6-3 cells were transfected with HA-LacI expression vector for 24 h using Lipofectamine 2000 reagent (Invitrogen) and then double immunostained with the indicated antibodies, followed by DAPI staining. Representative images and colocalization frequencies (%) of DDR-protein foci with LacI foci are shown. Values were calculated from the sum scores of at least two independent experiments. Scale bars, 10 µm. (B) U2OS 2–6-3 cells were transfected with the empty or the HA-LacI expression vectors for 24 h and then subjected to ChIP–quantitative PCR analysis with the indicated antibodies. Quantitative PCR was performed with primer pairs to detect lacO sequences. Results are shown as the percentage of input DNA. Means ± SD are shown ( n = 4). *, P < 0.05; **, P < 0.01; n.s., not significant (two-tailed Student’s t test). (C and D) U2OS 2–6-3 cells transfected as in (B) were treated with 10 µM VE-821 (ATR/ATM inhibitor), 10 µM KU-55933 (ATM inhibitor), or a control vehicle (DMSO) for 6 h and then subjected to immunoblotting. Data are representative of two or more independent experiments. In this experimental condition, VE-821 selectively inhibited ATR-mediated phosphorylation of Chk1 (Ser345), but not autophosphorylation of ATM (Ser1981). CBB, Coomassie Brilliant Blue; FANCD2-Ub, monoubiquitinated FANCD2.
    Figure Legend Snippet: Recruitment of various DDR proteins to lacO arrays after transient expression of HA-LacI in U2OS 2–6-3 cells. (A) U2OS 2–6-3 cells were transfected with HA-LacI expression vector for 24 h using Lipofectamine 2000 reagent (Invitrogen) and then double immunostained with the indicated antibodies, followed by DAPI staining. Representative images and colocalization frequencies (%) of DDR-protein foci with LacI foci are shown. Values were calculated from the sum scores of at least two independent experiments. Scale bars, 10 µm. (B) U2OS 2–6-3 cells were transfected with the empty or the HA-LacI expression vectors for 24 h and then subjected to ChIP–quantitative PCR analysis with the indicated antibodies. Quantitative PCR was performed with primer pairs to detect lacO sequences. Results are shown as the percentage of input DNA. Means ± SD are shown ( n = 4). *, P < 0.05; **, P < 0.01; n.s., not significant (two-tailed Student’s t test). (C and D) U2OS 2–6-3 cells transfected as in (B) were treated with 10 µM VE-821 (ATR/ATM inhibitor), 10 µM KU-55933 (ATM inhibitor), or a control vehicle (DMSO) for 6 h and then subjected to immunoblotting. Data are representative of two or more independent experiments. In this experimental condition, VE-821 selectively inhibited ATR-mediated phosphorylation of Chk1 (Ser345), but not autophosphorylation of ATM (Ser1981). CBB, Coomassie Brilliant Blue; FANCD2-Ub, monoubiquitinated FANCD2.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot

    Silencing of SLX4, XPF, MUS81, and FANCD2, and additional data for SLX4- and FANCD2-mediated accumulation of the DDR factors at the LacI-bound lacO . (A and B) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3 or siSLX4-4)–targeting siRNAs for 48 h. (A) Confirmation of SLX4 knockdown. Cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Coomassie Brilliant Blue staining served as a loading control. (B) After siRNA treatment, cells were further treated with 1 µM 4-OHT for 2 h and then subjected to colocalization analysis as described in . Values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–F) Cells were transfected with control siRNAs (a mixture of siGFP and siLuci) or siRNAs targeting XPF (siXPF-1 and siXPF-2) or MUS81 (siMUS81-1 or siMUS81-2) for 48 h and analyzed as described in A and B. ***, P < 0.001; n.s., not significant (χ 2 test). (G and H) Cells were transfected with control (mixture of siGFP and siLuci) or FANCD2 (siFANCD2-1 or siFANCD2-2)-targeting siRNAs for 48 h. (G) Cells were then subjected to immunoblotting with anti-FANCD2 antibody. (H) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then subjected to colocalization analysis as described in B. **, P < 0.01; ***, P < 0.001 (χ 2 test). (I) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or a vehicle (DMSO) for 6 h and 1 µM 4-OHT for the last 2 h (as in ). Cells were then subjected to SDS-PAGE and immunoblotting to confirm that ATR-mediated Chk1 phosphorylation is inhibited by VE-821 treatment. (J) U2OS 40–2-6 ER-LacI cells were incubated with 1 µM 4-OHT and either DMSO as a vehicle or 50 µM mirin for 2 h. Colocalization frequencies of RPA foci with LacI foci are analyzed as described in B. n.s., not significant (χ 2 test). (K) U2OS 40–2-6 ER-LacI cells were transfected with the indicated siRNAs. At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for a further 6 h and then analyzed as described in B. ***, P < 0.001 (χ 2 test).
    Figure Legend Snippet: Silencing of SLX4, XPF, MUS81, and FANCD2, and additional data for SLX4- and FANCD2-mediated accumulation of the DDR factors at the LacI-bound lacO . (A and B) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3 or siSLX4-4)–targeting siRNAs for 48 h. (A) Confirmation of SLX4 knockdown. Cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Coomassie Brilliant Blue staining served as a loading control. (B) After siRNA treatment, cells were further treated with 1 µM 4-OHT for 2 h and then subjected to colocalization analysis as described in . Values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–F) Cells were transfected with control siRNAs (a mixture of siGFP and siLuci) or siRNAs targeting XPF (siXPF-1 and siXPF-2) or MUS81 (siMUS81-1 or siMUS81-2) for 48 h and analyzed as described in A and B. ***, P < 0.001; n.s., not significant (χ 2 test). (G and H) Cells were transfected with control (mixture of siGFP and siLuci) or FANCD2 (siFANCD2-1 or siFANCD2-2)-targeting siRNAs for 48 h. (G) Cells were then subjected to immunoblotting with anti-FANCD2 antibody. (H) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then subjected to colocalization analysis as described in B. **, P < 0.01; ***, P < 0.001 (χ 2 test). (I) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or a vehicle (DMSO) for 6 h and 1 µM 4-OHT for the last 2 h (as in ). Cells were then subjected to SDS-PAGE and immunoblotting to confirm that ATR-mediated Chk1 phosphorylation is inhibited by VE-821 treatment. (J) U2OS 40–2-6 ER-LacI cells were incubated with 1 µM 4-OHT and either DMSO as a vehicle or 50 µM mirin for 2 h. Colocalization frequencies of RPA foci with LacI foci are analyzed as described in B. n.s., not significant (χ 2 test). (K) U2OS 40–2-6 ER-LacI cells were transfected with the indicated siRNAs. At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for a further 6 h and then analyzed as described in B. ***, P < 0.001 (χ 2 test).

    Techniques Used: Transfection, SDS Page, Western Blot, Staining, Incubation

    Silencing of SLX4–XPF suppresses accumulation of DDR factors at lacO , and the subsequent accumulations of ATR and FANCD2 are interdependent. (A) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3)-targeting siRNAs for 48 h. The cells were further treated with 1 µM 4-OH-tamoxifen (4-OHT) for 2 h, then subjected to colocalization analysis by double immunostaining. Similar experiments using other siRNAs are shown in . (B–D) U2OS 40–2-6 ER-LacI cells were transfected with control (mixture of siGFP and siLuci) siRNAs or siRNAs targeting XPF (siXPF-1 and siXPF-2), MUS81 (siMUS81-2), or FANCD2 (siFANCD2-2) for 48 h. (B and C) Following siRNA treatment, cells were treated with 1 µM 4-OHT for 2 h and subjected to colocalization analysis. (D) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then analyzed. Similar experiments using other siRNAs are shown in . (E) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or vehicle only (DMSO) for 6 h, with 1 µM 4-OHT for the last 2 h, followed by colocalization analysis. For A and B, the means ± SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test). For C–E, the values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also depicted.
    Figure Legend Snippet: Silencing of SLX4–XPF suppresses accumulation of DDR factors at lacO , and the subsequent accumulations of ATR and FANCD2 are interdependent. (A) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3)-targeting siRNAs for 48 h. The cells were further treated with 1 µM 4-OH-tamoxifen (4-OHT) for 2 h, then subjected to colocalization analysis by double immunostaining. Similar experiments using other siRNAs are shown in . (B–D) U2OS 40–2-6 ER-LacI cells were transfected with control (mixture of siGFP and siLuci) siRNAs or siRNAs targeting XPF (siXPF-1 and siXPF-2), MUS81 (siMUS81-2), or FANCD2 (siFANCD2-2) for 48 h. (B and C) Following siRNA treatment, cells were treated with 1 µM 4-OHT for 2 h and subjected to colocalization analysis. (D) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then analyzed. Similar experiments using other siRNAs are shown in . (E) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or vehicle only (DMSO) for 6 h, with 1 µM 4-OHT for the last 2 h, followed by colocalization analysis. For A and B, the means ± SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test). For C–E, the values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also depicted.

    Techniques Used: Transfection, Double Immunostaining, Two Tailed Test

    FANCD2 and MUS81 accumulate on the lacO array in mitosis, where MiDAS is activated, and LacI binding during S phase increases FANCD2 colocalization with lacO . (A) Experimental scheme. U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase with 7 µM RO-3306 treatment for 20 h and 1 µM 4-OHT for 20 h or the last 4 h. G2-arrested cells were then released into fresh medium for 5 min and subjected to double immunostaining, DAPI staining, and analysis. (B) Confirmation of G2-phase synchronization with RO-3306. U2OS 40–2-6 ER-LacI cells synchronized in G2 phase by incubation with 7 µM RO-3306 for 16 h were subjected to FACS analysis. (C and D) Representative images (left) and colocalization frequencies (right) are shown. Values were calculated from the sum scores of two independent experiments. ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also shown. Yellow arrows indicate colocalization of FANCD2 (C) or MUS81 (D) with the LacI foci. Scale bars, 10 µm. (E) Experimental workflow for MiDAS detection. U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase as described in A. The G2-arrested cells were released or left unreleased, in each case for 20 min in the presence of 10 µM BrdU. DNA was denatured by treatment with HCl for detection of DNA labeled with BrdU. Cells were then double immunostained with anti-LacI and anti-BrdU antibodies, followed by DAPI staining and analysis. (F) Representative images of G2-arrested cells or prophase/prometaphase cells with LacI foci. BrdU incorporation (indicating MiDAS) was detected specifically in mitotic cells (yellow arrows), but not in G2 cells (white arrows). Scale bar, 10 µm. (G) The graph indicates colocalization frequencies of BrdU foci with LacI foci in G2 phase and prophase/prometaphase cells. Values were calculated from the sum scores of two independent experiments. *, P < 0.05; ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also shown.
    Figure Legend Snippet: FANCD2 and MUS81 accumulate on the lacO array in mitosis, where MiDAS is activated, and LacI binding during S phase increases FANCD2 colocalization with lacO . (A) Experimental scheme. U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase with 7 µM RO-3306 treatment for 20 h and 1 µM 4-OHT for 20 h or the last 4 h. G2-arrested cells were then released into fresh medium for 5 min and subjected to double immunostaining, DAPI staining, and analysis. (B) Confirmation of G2-phase synchronization with RO-3306. U2OS 40–2-6 ER-LacI cells synchronized in G2 phase by incubation with 7 µM RO-3306 for 16 h were subjected to FACS analysis. (C and D) Representative images (left) and colocalization frequencies (right) are shown. Values were calculated from the sum scores of two independent experiments. ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also shown. Yellow arrows indicate colocalization of FANCD2 (C) or MUS81 (D) with the LacI foci. Scale bars, 10 µm. (E) Experimental workflow for MiDAS detection. U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase as described in A. The G2-arrested cells were released or left unreleased, in each case for 20 min in the presence of 10 µM BrdU. DNA was denatured by treatment with HCl for detection of DNA labeled with BrdU. Cells were then double immunostained with anti-LacI and anti-BrdU antibodies, followed by DAPI staining and analysis. (F) Representative images of G2-arrested cells or prophase/prometaphase cells with LacI foci. BrdU incorporation (indicating MiDAS) was detected specifically in mitotic cells (yellow arrows), but not in G2 cells (white arrows). Scale bar, 10 µm. (G) The graph indicates colocalization frequencies of BrdU foci with LacI foci in G2 phase and prophase/prometaphase cells. Values were calculated from the sum scores of two independent experiments. *, P < 0.05; ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also shown.

    Techniques Used: Binding Assay, Double Immunostaining, Staining, Incubation, Labeling, BrdU Incorporation Assay

    A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).
    Figure Legend Snippet: A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).

    Techniques Used: Binding Assay, Generated, Inhibition

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    Cell Signaling Technology Inc fancd2
    ( 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 <t>FANCD2</t> 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.
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    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 <t>FANCD2</t> 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
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    A. Immunofluorescence detection of <t>FANCD2</t> 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).
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    ( 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.

    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

    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

    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

    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).

    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