Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Depletion of FANCM induces replication stress. U2-OS cells were transfected with siRNA targeting Luciferase (Luc) or two different siRNA targeting FANCM (FANCM and FANCM-U). Cells were then stained with antibodies recognizing γH2AX or Chk1-pS345 (A and B, labeled as pChk1), RPA32-pS4S8 (C and D), or BLM (E and F). All nuclei were also stained with DAPI. More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments. Standard two-sided t test: ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Chk1-pS345 foci colocalize with the BLM and PML foci. (A) U2-OS cells were transfected with siRNA targeting either Luciferase (Luc) or two different siRNA against FANCM (labeled as M and MU). Cell lysates were prepared and blotted with antibodies as indicated on the right. Asterisks indicate cross-reaction bands. The vertical line marks the phosphorylated RPA32. (B) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), CtIP, Mre11, FANCM (FANCM-U), or in combination. Cells were then costained with antibodies recognizing TRF1 or RPA32-pS4S8. More than 200 cells were counted for each sample. All error bars are SD obtained from two different experiments. (C and D) U2-OS cells were transfected with siRNA targeting either Luciferase (Luc) or two different siRNA against FANCM (labeled as FANCM and FANCM-U). Cells were then costained with antibodies recognizing Chk1-pS345 (labeled as pChk1) and BLM (C) or Chk1-pS345 and PML (D). All nuclei were also stained with DAPI. Standard two-sided t test: ***P < 0.001. n.s., not significant.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Depletion of FANCM induces replication stress at ALT telomeres. U2-OS cells were transfected with siRNA targeting Luciferase (Luc) or two different siRNA targeting FANCM (FANCM or M and FANCM-U or MU). (A–C and G) siRNA transfected cells were costained with an antibody recognizing Chk1-pS345 (labeled as pChk1) together with a PNA probe recognizing the G-rich strand of telomeres (A, TelC), an antibody recognizing TRF1 (B), or TRF2 (C). (D–F and H) siRNA transfected cells were costained with an antibody recognizing BLM, together with TelC (D), an antibody recognizing TRF1 (E), or TRF2 (F). All nuclei were also stained with DAPI. More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments. Standard two-sided t test: ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Depletion of FAAP24 induces replication stress at the ALT telomeres. U2-OS cells were transfected with siRNA targeting Luciferase (Luc) or two different siRNA targeting FAAP24 (FAAP24-1 and -2). Cells were then costained with an antibody recognizing Chk1-pS345 (labeled as pChk1, A–C) or BLM (D–F) together with a PNA probe recognizing the G-rich strand of telomeres (TelC) or an antibody recognizing TRF1. All nuclei were stained with DAPI. More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments. Standard two-sided t test: ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Depletion of MHF1, MHF2, or MHF1 and -2 induces replication stress at the ALT telomeres. U2-OS cells were transfected with siRNA targeting Luciferase (Luc), MHF1, MHF2, or MHF1 and -2. Cells were then costained with an antibody recognizing Chk1-pS345 (labeled as pChk1, A–C) or BLM (D–F), together with a PNA probe recognizing the G-rich strand of telomeres (TelC) or an antibody recognizing TRF1. All nuclei were stained with DAPI. More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments. Standard two-sided t test: ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Depletion of the component of the FANCM-FAAP24-MHF1/2 complex individually or in combination induces replication stress at the telomeres. U2-OS cells were transfected with siRNA as indicated. Cells were then costained with an antibody recognizing TRF1 together with an antibody recognizing Chk1-pS345 (A), RPA32-pS4S8 (B), or BLM (C). More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: The effect of different FA proteins on the Chk1-pS345 and BLM foci formation. U2-OS cells were transfected with siRNA as indicated. Cells were costained with an antibody recognizing TRF1 together with an antibody recognizing Chk1-pS345 (A) or BLM (B). All nuclei were stained with DAPI. More than 200 cells were counted for each sample. All error bars are SD obtained from three different experiments.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: BLM promotes DNA end resection and DNA repair at the ALT telomeres. (A and B) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), BLM (BLM-1), FANCM (MU), or both BLM and FANCM (BLM-1+MU). Cell lysates were blotted with antibodies as indicated on the right (A). The siRNA transfected cells were also costained with antibodies recognizing either RPA32-pS4S8 or TRF1 (B). More than 200 cells were counted for each sample, and the percentage of cells with both RPA32-pS4S8 and TRF1 foci was plotted. (C and D) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), two different siRNA targeting BLM (BLM-1 and BLM-3), FANCM (MU), or both BLM and FANCM. The nucleus of siRNA transfected U2-OS cells was stained with DAPI. More than 200 cell nuclei were analyzed, and the percentage of cells with micronuclei was plotted (C). The viability of the same siRNA transfected cells in C was analyzed by crystal violet assay as detailed in Materials and Methods (D). All error bars are SD and obtained from three independent experiments. Standard two-sided t test: **P < 0.01.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Staining, Crystal Violet Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: The molecular mechanism behind the recruitment of BLM and BRCA1 to ALT telomeres. (A–C) BRCA1 localized to ALT telomeres. U2-OS cells (A and B) or Saos-2 cells (C) were transfected with siRNA targeting either Luciferase control (Luc) or two different siRNA targeting FANCM (M and MU). Cells were then costained with an antibody recognizing BRCA1 together with a PNA probe recognizing the telomere (A and C, TelC) or an antibody recognizing TRF1 (B). (D) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), BLM (BLM1), FANCM (MU), or both BLM and FANCM (BLM1+MU). Cells were then transfected with either GFP or GFP-BLM that is resistant to the BLM1 siRNA and costained with antibodies recognizing either BRCA1 or TRF1. (E) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), BRCA1 (BU), FANCM (MU), or both BRCA1 and FANCM (BU+MU). Cells were then transfected with either Vector or BRCA1 ORF and costained with antibodies recognizing either BLM or TRF1. (F–H) U2-OS cells were transfected with different siRNAs and then costained with antibodies recognizing TRF1, BRCA1 (F and G), or BLM (H). More than 200 cells were counted for each sample. All error bars are SD and obtained from two independent experiments. Standard two-sided t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Transfection, Luciferase, Control, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: BRCA1 and BLM cooperatively promotes DNA end resection and homologous recombination at ALT telomeres. (A) U2-OS cells were transfected with siRNA targeting Luciferase (Luc), BRCA1 (BU), FANCM (MU), or both BRCA1 and FANCM (BU+MU). Cell lysates were blotted with antibodies as indicated on the right. The siRNA transfected cells were also costained with antibodies recognizing either RPA32-pS4S8 or TRF1 (B). (C) The nuclei of siRNA transfected U2-OS cells were stained with DAPI. More than 200 nuclei were analyzed, and the percentage of cells with micronuclei was plotted. (D and E) The viability of the siRNA transfected cells was analyzed by crystal violet assay as detailed in Materials and Methods. (F and G) U2-OS cells were transfected with siRNA targeting either Luciferase (Luc) or two different siRNA targeting FANCM (FANCM and FANCM-U). Cells were then costained with antibodies recognizing Rad51 or TRF1. All nuclei were also stained with DAPI. (H) U2-OS cells were transfected with different siRNA and then costained with antibodies recognizing either Rad51 or TRF1. More than 200 cells were counted for each sample. All error bars are SD and obtained from three independent experiments. Standard two-sided t test: ***P < 0.001.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Homologous Recombination, Transfection, Luciferase, Staining, Crystal Violet Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

doi: 10.1073/pnas.1708065114

Figure Lengend Snippet: Knockdown efficiency of the siRNAs targeting FAAP24, MHF1, MHF2, BLM, BRCA1, or Abra1. U2-OS cells were transfected with siRNA targeting Luciferase (Luc), two different siRNA targeting FAAP24 (FAAP24-1 and -2) (A), MHF1, MHF2, or both MHF1 and MHF2 (MHF1/2) (B), two different siRNA targeting BLM (BLM-1 and -3) (C), two different siRNA targeting BRCA1 (BRCA1-A and -U) (D), or Abra1 (E). Cell lysates were prepared and blotted with antibodies as indicated on the right. (F) A diagram of how ALT cells suppress FANCM deficiency-induced replication stress at telomeres.

Article Snippet: Antibodies used for immunofluorescent staining include Chk1-pS345 (Cell Signaling, 2348); RAP32-pS4S8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A and A300-120A); BRCA1 (Bethyl, A301-377A); anti–γ-H2AX (Millipore, 05–636) and (Bethyl, A300-081A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); and TRF2 (Millipore, 05–521).

Techniques: Knockdown, Transfection, Luciferase

Journal: Nature Communications

Article Title: Bloom helicase mediates formation of large single–stranded DNA loops during DNA end processing

doi: 10.1038/s41467-022-29937-7

Figure Lengend Snippet: a Western blots of extracts from U2OS (SA-GFP and DR-GFP) cells depleted for BLM and EXO1 were reconstituted with the indicated genotypes and immune-stained with anti-BLM and anti-Tubulin antisera, as indicated. b Bar graph showing results for the SA-GFP reporter assay in U2OS cells with the indicated genotypes; data points represent %SSA. c Bar graph showing results for the DR-GFP reporter assay in U2OS cells with the indicated genotypes; data points represent %HR. In b and c data points reflect the mean ± SEM for N = three independent experiments.

Article Snippet: NC membranes were blocked using 5% dry milk in TBST (Tris-buffered saline with 0.1% Tween® 20 detergent) and stained with primary antibodies: anti-BLM (Santa Cruz Biotechnology, Cat. No.: SC-365753; used at a 1:2000 dilution; or Bethyl, Cat. No.: A300-100A; used at a 1:1000 dilution), anti-EXO1(Bethyl, Cat. No.: A302-639A; used at a 1:1000 dilution), anti-FLAG (M2) (Sigma Cat. No.: F1804; used at a 1:1000 dilution), anti-Actin (CST, Cat. No.: 12262S; used at a 1:3000 dilution), anti-GFP (Invitrogen, Cat. No.: A-6455; used at a 1:4000 dilution), or Alpha–Tubulin–HRP (Cell Signaling Technology, Cat. No.: CST 11H10; used at a 1:5000 dilution), as indicated, O/N at 4 °C, washed with TBST buffer 3 times for 10 min each and incubated with appropriate secondary antibody (anti-mouse IgG HRP, Invitrogen Cat. No.: A16078; used at a 1:4000 dilution; or anti-rabbit IgG Fc HRP, Cat. No.: A16116; used at a 1:4000 dilution) for 1 h at RT.

Techniques: Western Blot, Staining, Reporter Assay