Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used: RAD51 ([N1C2] GeneTex), RPA32 (19/NA18 CalBiochem), RPA32 (4E4 Cell Signaling), CtIP (271339 Santa Cruz), RIF1 (A300–569A Bethyl), 53BP1 (3802 Millipore), phospho53BP1 (3428 Cell Signaling), BRCA1 (6954 Santa Cruz) BRCA1 (07–434 Millipore), PCNA (sc-56 Santa Cruz) and, HA tag (Cell Signaling 1:500).

Techniques: Recombinant, Plasmid Preparation, Staining, Mutagenesis, Modification, Software

Journal: Journal of Cell Science

Article Title: RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner

doi: 10.1242/jcs.240036

Figure Lengend Snippet: RIF1 inhibits HRR after exposure to high IR doses but not after low IR doses. (A) Restoration of radiation-induced HRR inhibition by RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were analyzed with the DR-GFP assay as in Fig. 1 B. (B,C) Restoration of 3 Gy-induced RAD51 foci by RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were stained for RAD51 focus formation 1 h after exposure to 0.5 Gy or 3 Gy and the number of RAD51 foci per nucleus quantified. CENPF was used as a marker of the S/G2 cell cycle phases. Nuclear boundaries are marked with dotted lines and boxes indicate regions shown in magnified views. Scale bar: 10 μm. (D) RPA2 focus formation at DSB sites. HeLa cells were fixed 1 h after irradiation and were stained with anti-RPA2 antibodies. 53BP1 foci were used as markers of DSB sites. (E) Increased phosphorylation of RPA2 at Ser4 or Ser8 (pS4/S8) after RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were irradiated with 3 Gy and collected 1 h after irradiation, for sample preparation. RPA2pS4/S8 and RPA2 proteins were detected by western blotting. Data in A,C and D are mean±s.d. Indicated P values were calculated using a two-way ANOVA, followed by Tukey's multiple comparisons test.

Article Snippet: The primary antibodies used for immunofluorescence were RAD51 (B01P; Abnova; 1:1000), RPA2 (Ab-2; Calbiochem; 1:200), RIF1 (A300-569A; Bethyl Laboratories Inc.; 1:1000), 53BP1 (MAB3802, Merck Millipore or A300-273A, Bethyl Laboratories Inc.; 1:1000), phospho-53BP1 T543 (3428; Cell Signaling Technology; 1:1000), MCM2 (D7G11; Cell Signaling Technology; 1:1000) and CENPF (ab5; Abcam; 1:2000).

Techniques: Inhibition, Transfection, Staining, Marker, Irradiation, Sample Prep, Western Blot

Journal: Journal of Cell Science

Article Title: RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner

doi: 10.1242/jcs.240036

Figure Lengend Snippet: BRCA1 antagonizes ATM-dependent RIF1 accumulation at low IR doses. (A,B) Marginal formation of 0.5 Gy-induced foci of RIF1 and phospho-53BP1 in S-phase cells. HeLa cells were synchronized with a double thymidine block in G1 or S phases and were irradiated with 0.5 Gy or 3 Gy of IR. Cells were fixed and stained with anti-RIF1 (A) or anti-phospho-53BP1 (B) antibodies 0.5 h after irradiation and the number of foci per nucleus was quantified. (C) Phosphorylation of 53BP1 and CHK2 after irradiation. HeLa cells synchronized in G1 or S phase were irradiated and collected 1 h after irradiation for western blotting with the indicated antibodies. β-actin is shown as a loading control. (D,E) Phospho-53BP1 (D) or RIF1 (E) foci formation after irradiation in BRCA1-depleted and BRCA1-overexpressing cells. HeLa cells transfected with siCtrl, siBRCA1 or a BRCA1-expressing vector were stained with anti-phospho-53BP1 or anti-RIF1 antibodies 0.5 h after IR exposure of 0.5 Gy or 3 Gy. (F) Enhanced RAD51 focus formation after exposure to 3 Gy with BRCA1 overexpression. HeLa cells transfected with empty or BRCA1-expressing vectors were stained for RAD51 focus formation 1 h after exposure to 0.5 Gy or 3 Gy. Data in A,B,D–F are mean±s.d., and the P values indicated were calculated by two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: The primary antibodies used for immunofluorescence were RAD51 (B01P; Abnova; 1:1000), RPA2 (Ab-2; Calbiochem; 1:200), RIF1 (A300-569A; Bethyl Laboratories Inc.; 1:1000), 53BP1 (MAB3802, Merck Millipore or A300-273A, Bethyl Laboratories Inc.; 1:1000), phospho-53BP1 T543 (3428; Cell Signaling Technology; 1:1000), MCM2 (D7G11; Cell Signaling Technology; 1:1000) and CENPF (ab5; Abcam; 1:2000).

Techniques: Blocking Assay, Irradiation, Staining, Western Blot, Transfection, Expressing, Plasmid Preparation, Over Expression

Journal: Journal of Cell Science

Article Title: RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner

doi: 10.1242/jcs.240036

Figure Lengend Snippet: RIF1 inhibits replication initiation after exposure to high IR doses but not after low IR doses. (A) DNA synthesis after exposure to 3 Gy IR in siCtrl and siRIF1 cells. Incorporation of EdU was measured 1 h after irradiation using a flow cytometer. (B) Schematic illustration of pulse-labeling and PCNA staining. HeLa cells synchronized in mid-S phase were pulse-labeled with EdU and stained with anti-PCNA antibodies after exposure to 0.5 Gy or 3 Gy of IR. (C,D) Time course of PCNA and EdU foci formation in non-irradiated HeLa cells. PCNA foci, scored as either colocalizing with EdU foci (>0.05 µm 2 overlap area, PCNA with EdU) or not (<0.05 µm 2 overlap area, PCNA w/o EdU), were quantified in each nucleus. Data are the mean±s.d. Boxes in C indicate regions shown in magnified images. Scale bar: 10 μm. (E) Transient replication block after exposure to 3 Gy of IR. After pulse-labeling, HeLa cells were irradiated with 0.5 Gy or 3 Gy and were fixed 1 or 3 h after IR exposure. Cells were stained for EdU and PCNA foci formation. PCNA foci without EdU foci were counted. (F) Restoration of the replication block after RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were irradiated with 3 Gy and were fixed 1 h after IR exposure. PCNA foci without EdU foci were counted. Data are the mean±s.d. of three independent experiments. P values indicated were calculated using a Chi-squared test. (G,H) DNA synthesis at DSB sites was measured by the colocalization of EdU with 53BP1. HeLa cells transfected with either siCtrl or siRIF1 were irradiated with 3 Gy and fixed 1 h after IR exposure. Cells were stained for EdU and 53BP1 foci formation. Frequency of 53BP1 foci colocalized with EdU were measured. Data in H are mean±s.d. and the P value indicated was calculated using a Student's t -test. Scale bar: 10 μm.

Article Snippet: The primary antibodies used for immunofluorescence were RAD51 (B01P; Abnova; 1:1000), RPA2 (Ab-2; Calbiochem; 1:200), RIF1 (A300-569A; Bethyl Laboratories Inc.; 1:1000), 53BP1 (MAB3802, Merck Millipore or A300-273A, Bethyl Laboratories Inc.; 1:1000), phospho-53BP1 T543 (3428; Cell Signaling Technology; 1:1000), MCM2 (D7G11; Cell Signaling Technology; 1:1000) and CENPF (ab5; Abcam; 1:2000).

Techniques: DNA Synthesis, Irradiation, Flow Cytometry, Labeling, Staining, Blocking Assay, Transfection

Journal: eLife

Article Title: Mycobacterium tuberculosis exploits host ATM kinase for survival advantage through SecA2 secretome

doi: 10.7554/eLife.51466

Figure Lengend Snippet: ( a ) Representative images of immunofluorescence showing RPA2 foci (AlexaFluor594-Red) in RAW264.7 macrophages infected with GFP expressing Rv, Ra (Green) at 24 h p.i. ( b and c ) We assessed the activation of various DDR proteins activated in response to DSBs mediated through Rv infection in THP-1 ( b ) and RAW264.7 ( c ) cells. WCL prepared at indicated h.p.i. were subjected to immunoblotting with α-γH2AX, α-pATM(S1981), α-pCHK2(T68), α-p53BP1-T543, α-MRE11, α-pNbs1-S343, α−53BP1, α-pMDC1, α-pMDC1-T4 and α-β-actin antibodies.

Article Snippet: All cell culture reagents were procured from Thermo fisher scientific Inc or Hyclone. α-γH2AX (2577S), α-ATM (2873S), α-pATR-S428 (2853S), α-pChk1-S345 (2348S), α-pChk2-T68 (2661S), α-Chk2 (2662S), α-CHK1(2360), α-ATR (13934), α-pAkt-S473 (9271S), α-Akt (9272S), α-p53BP1-T543 (3428S), α-MRE11(4895S), α-pNbs1-S343 and α-Nbs1 (3002S) antibodies were procured from Cell Signalling Technologies. α-pATM-S1891(ab81292) α−53BP1 (ab36823), α-pMDC1-T4 (ab35967), α-RPA2 (ab2175) and α-p53 (ab28) antibody was procured from Abcam. α-p21 (sc-817), α-β-actin (sc-47778), α-β-tubulin (sc-55529) and α-GAPDH (sc-25778) antibodies were purchase from Santacruz Biotechnology. α-DNA-PKcs (SAB4502385), α-pDNA-PKcs-S2056 (SAB4504169) were purchased from Sigma. α-MDC1 and α-pRPA2-S4/S8 was purchased from Novus Biologicals and HRP conjugated secondary antibodies were purchased from Jackson laboratories. α-MDC1 was purchased from Novus Biologicals.

Techniques: Immunofluorescence, Infection, Expressing, Activation Assay, Western Blot

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 plays role in DNA double-strand break repair in S-phase cells. ( A ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i and two independent WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with 53BP1 antibody. Click chemistry was used to visualize EdU. Mean of median foci number +/- SD is plotted (n ≥ 3). Statistical significance evaluated by two tailed t -test. ( B ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. As in A. ( C ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( D ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( E ) Traffic light reporter assay in U2OS cells after transfection with indicated siRNA. Cells were transfected with ISceI together with BFP-donor vector with or without pretreatment with 1 μM WIP1i 2 days after siRNA transfection. Efficiency of repair was analyzed by FACS 3 days after ISceI and BFP-donor transfection. Plotted is mean +/− SD. Statistical significance evaluated by two-tailed t -test. ( F ) Efficiency of repair by HR and NHEJ in Traffic light reporter assay as in E. ( G ) Representative plots from Traffic light reporter assay in E.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Irradiation, Knock-Out, Labeling, Staining, Two Tailed Test, Mutagenesis, Reporter Assay, Transfection, Plasmid Preparation

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 delays recruitment of BRCA1 and dephosphorylation of 53BP1 at T543. ( A ) Co-immunoprecipitation of WIP1 and 53BP1. HEK293 cells were transfected with either empty GFP or GFP-WIP1, subjected to immunoprecipitation using GFP-Trap 24 h after transfection and by Western blotting with 53BP1 antibody. Ponceau staining with indicated positions of GFP (empty arrowhead) and GFP-WIP1 (full arrowhead) are shown. ( B ) HEK293 cells transfected with EGFP or EGFP-WIP1 were exposed to 3 Gy of IR, collected at indicated times and proteins were immunoprecipitated by GFP Trap. ( C ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points after IR and stained with p53BP1 T543 antibody. Click chemistry was used to visualize EdU. Mean of median total intensity +/− SD is plotted. ( D ) Western blot analysis of whole cell lysates of U2OS cells transfected with GAPDH or PP4C siRNA in response to irradiation and/or WIP1 inhibitor. ( E ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were transfected with control or PP4C siRNA 2 days before irradiation. Cells were processed and analyzed as in C. ( F ) Quantification of RPA2 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i were pulse-labeled with EdU for 30 minutes before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with RPA2 and RAD51 antibodies. Click chemistry was used to visualize EdU. Mean of median foci number +/− SD is plotted. ( G ) Quantification of RAD51 foci in replicating (EdU+) cells after irradiation as in F.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Transfection, Western Blot, Staining, Irradiation, Knock-Out, Labeling

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 deficient cells are more sensitive to PARP inhibition. ( A ) Cell survival of parental U2OS, two independent U2OS-WIP1-KO cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD, n ≥ 3. Statistical significance evaluated by two-way ANOVA. ( B ) Cell survival of parental U2OS, U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 in response to 5 μM olaparib as in A. Statistical significance evaluated by two-tailed t -test (n ≥ 3). ( C ) Percentage of dead cells was evaluated by Hoechst 33258 staining and FACS analysis 7 days after treatment with 5 μM olaparib in U2OS cell line with or without combined treatment with WIP1i. Plotted is mean +/− SD. ( D ) Cell survival of RPE and MCF7 cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD. N ≥ 3. Statistical significance evaluated by two-way ANOVA. ( E ) Cells were transfected with control siRNA (siNC) or siRNA to PP4C (siPP4C). Cell survival was evaluated after 7 days of treatment with olaparib and DMSO or WIP inhibitor. Statistical significance evaluated by two-tailed t -test (n = 3). ( F ) Quantification of 53BP1 foci number 3 days after treatment with olaparib. U2OS cells were treated with indicated doses of olaparib together with or without WIP1i for 3 days, fixed, stained with 53BP1 antibody and percentage of cells having 0–3, 3–10 and >10 foci were quantified. Mean +/− SD is plotted, n ≥ 3. ( G ) Quantification of 53BP1 foci after treatment with olaparib. U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were treated with WIP1i and olaparib for 3 days, fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted, n ≥ 3. Statistical significance evaluated by two-tailed t -test. ( H ) Response of U2OS and U2OS-WIP1-KO cell lines to treatment with 5 μM olaparib for 24–72h was analyzed by Western blotting using indicated antibodies. I ) Quantification of 53BP1 foci 3 days after treatment with olaparib. MCF7 cells were transfected with indicated siRNAs and treated after 2 days with WIP1i and olaparib alone or combined for further 3 days. Cells were fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted. Statistical significance evaluated by two-tailed t -test.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Inhibition, Viability Assay, Mutagenesis, Two Tailed Test, Staining, Transfection, Western Blot