Journal: Genes & Development

Article Title: ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

doi: 10.1101/gad.292516.116

Figure Lengend Snippet: ZMYM3 antagonizes the BRCA1-A complex to promote HR. ( A ) ZMYM3 promotes HR and opposes RAP80 and ABRA1 inhibition of HR. DR-GFP reporter assays were performed after depletion or codepletion of the indicated proteins by siRNAs. Data represent mean ± SD. n = 3. ( B ) Confirmation of knockdown efficiency by Western blotting from experiments performed in A . ( C ) 53BP1 depletion rescues HR defects in ZMYM3-depleted cells. Experiments were performed as in A . ( D ) ZMYM3 knockout cells are sensitive to IR and PARP inhibitors compared with parental U2OS cells. Cells were challenged with IR or PARP inhibitor as indicated and were analyzed by colony formation assays. Data represent mean ± SD. n = 3. ( E ) Western blotting analysis of knockdown efficiency in cells transfected with BRCA1 siRNA. ( F ) Epistasis analysis of ZMYM3 and BRCA1. Wild-type and ZMYM3 knockout cells either alone or in combination with siBRCA1 were challenged with IR and PARP inhibitor followed by colony formation assays. ( G ) Chromosome aberration analyses in ZMYM3 knockout and BRCA1 knockdown cells. Experiments were performed as in E. ( H ) Complementation assay of ZMYM3 knockout cells. ZMYM3 knockout cells with empty vector or wild-type ZMYM3 were analyzed as in D . (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t -test.

Article Snippet: Primary antibodies used in this study were Flag M2 (Sigma, F1804), Myc (Santa Cruz Biotechnology, SC-40), H2AX (Millipore, 07-627), γH2AX (Millipore, 05-636), H2AZ (Cell Signaling, 2718S), macroH2A (Abcam, AB37264), H2A.Bbd (Millipore, 06-1319), ZMYM3 (Abcam, AB106626), ATM (Santa Cruz Biotechnology, SC-135663), pATM S1981 (Abcam, AB81292), β-tubulin (Abcam, AB6046), RAD51 (Abcam, AB88572), RAP80 (Bethyl Laboratories, A300-763A), ABRA1 (Abcam, AB139191), BRCA1 (Santa Cruz Biotechnology, SC-6954), HRP-linked anti-GST (Sigma, A7340), MBP (Abcam, AB9084), phospho-H3 S10 (Cell Signaling, 3377), 53BP1 (Novus Biologicals, NB100-304), RAD18 (Cell Signaling, 9040), RPA2 (Abcam, AB2175), pRPA2 S33 (Bethyl Laboratories, A300-246), pRPA2 S4/S8 (Bethyl Laboratories, A300-245), Chk1 (Santa Cruz Biotechnology, SC-8408), and pChk1 (Cell Signaling, 2348).

Techniques: Inhibition, Knockdown, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Control

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-53BP1 , Novus Biologicals , Cat# NB100-904.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

Journal: Aging Cell

Article Title: Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

doi: 10.1111/acel.12838

Figure Lengend Snippet: Senescence‐associated secretory phenotype factors cause telomere dysfunction in a paracrine manner in cells that lack hTERT expression. (a) Percentage of early passage human BJ fibroblasts that immunostained positive for 1–2 (blue bars), 3 (red bars), 4–5 (green bars), or more than 5 (yellow) 53BP1 foci after a 48 hr incubation with conditioned medium from early passage cells (EP), senescent cells (Sen), hydroxyurea treated cells (HU), and zeocin treated cells (Zeo). Control cells (C) were untreated. Representative micrographs illustrating 53BP1 (green) foci are shown to the right. Nuclear DNA was counterstained with DAPI (blue). Scale bars: 20 µm. At least 100 cells were analyzed for each experiment. Error bars: ± SD . * p < 0.05 ( n = 4). (b) Percentage of cells that immunostained positive for telomere dysfunction‐induced DNA damage foci (TIF) treated as in (a). Representative micrographs of TIF‐positive cells and telomere (red)‐53BP1 (green) colocalizations for each group are shown to the right. Error bars: ± SD . * p < 0.05. (c) Percentage of normal human BJ fibroblasts (−hTERT; left graph) and BJ fibroblasts overexpressing hTERT (+hTERT; right graph) positive for TIF as treated in B. Error bars: ± SD . * p < 0.05 ( n = 3)

Article Snippet: The sources and dilutions of antibodies used were as follows: α‐SMA (SIGMA‐Aldrich, 1:1,000), 53BP1 (polyclonal; Novus, Littleton, CO), β‐Tubulin, p53(S15), phospho‐SMAD3 (Cell Signaling, Danvers, MA); p‐ATM(S1981) (Abcam, Cambridge, MA); p21 (Santa Cruz, Dallas, TX), TRF2 (Novus, Littleton, CO; 1:500), p16 (Santa Cruz, CA; 1:200); Nox‐4 (Abcam, Cambridge, MA); macroH2A (kind gift from Dr. Peter Adams), Lamin B1 (Abcam; 1:1,000); Ras (BD Transduction Laboratories, San Jose, CA; 1:1,000); p53 (DO‐1, BD biosciences); γH2AX(S139) (Upstate, Chicago, IL; 1:1,000); γ‐Tubulin (Sigma, St Louis, MO; 1:5,000).

Techniques: Expressing, Incubation

Journal: Aging Cell

Article Title: Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

doi: 10.1111/acel.12838

Figure Lengend Snippet: TGF‐β1 causes telomere dysfunction and G1 DNA damage checkpoint activation in normal human fibroblasts. (a) Percentage of early passage BJ fibroblasts that were positive for TIF following treatment with conditioned medium collected from either early passage BJ fibroblasts (EP) or senescent cells (Sen.) in the absence or presence of SB431542, a pharmacological inhibitor of TGF‐β1 signaling for 48 hr (Sen+Ti). Error bars: ± SD ; ( n = 4). * p < 0.005. Representative micrographs of TIF‐positive cells and telomere (red)‐53BP1 (green) colocalizations (arrows) for Sen and Sen+Ti group are shown to the right. (b) Percentage of early passage human BJ fibroblasts (−hTERT) and BJ fibroblasts overexpressing hTERT (+hTERT) that immunostained positive for 1–2 (blue bars), 3 (red bars), 4–5 (green bars), or more than 5 (yellow) 53BP1 foci and that were either untreated as control (C) or treated with recombinant TGF‐β1 (10 ng/ml) for 48 hr. Error bars: ± SD . * p < 0.05; ns: not significant; ( n = 3). Representative micrographs illustrating 53BP1 (green) foci are shown to the right. Nuclear DNA was counterstained with DAPI (blue). Scale bars: 20 µm. (c) Percentage of early passage human BJ fibroblasts (−hTERT) and BJ fibroblasts overexpressing hTERT (+hTERT) that immunostained positive for TIF foci and that were treated as in A. Error bars: ± SD . * p < 0.005; ns: not significant, p > 0.05, ( n = 3). Representative micrographs of TIF‐positive cells and telomere (red)‐53BP1 (green) colocalizations for (indicated by numbers) are shown to the right. (d) Distribution of telomere lengths based on their signal intensities ( x ‐axis; arbitrary units). Top histogram: all telomeric signals in cells treated with (yellow bars) or without TGF‐β1 (blue bars) for 48 hr. Bottom histogram: telomeric signals associated with 53BP1 foci in cells treated with TGF‐β1 (red bars) for 48 hr. n = 1,948, no. of telomeric signals analyzed. Average telomere signal intensities, and percent reduction in response to TGF‐β1 treatment, are indicated. (e) Immunoblots of extracts from early passage BJ fibroblasts (−hTERT) and BJ fibroblasts overexpressing hTERT (+hTERT) that were either control treated (C) or treated with TGF‐β1 for 72 hr using indicated antibodies. β‐tubulin was used as loading control. (f) Proliferation curves of BJ fibroblasts (−hTERT; left graph) and BJ fibroblasts overexpressing hTERT (+hTERT; right graph) following treatment with TGF‐β1 (dark line) or control (light line). Error bars: ± SD , * p < 0.005, ( n = 3)

Article Snippet: The sources and dilutions of antibodies used were as follows: α‐SMA (SIGMA‐Aldrich, 1:1,000), 53BP1 (polyclonal; Novus, Littleton, CO), β‐Tubulin, p53(S15), phospho‐SMAD3 (Cell Signaling, Danvers, MA); p‐ATM(S1981) (Abcam, Cambridge, MA); p21 (Santa Cruz, Dallas, TX), TRF2 (Novus, Littleton, CO; 1:500), p16 (Santa Cruz, CA; 1:200); Nox‐4 (Abcam, Cambridge, MA); macroH2A (kind gift from Dr. Peter Adams), Lamin B1 (Abcam; 1:1,000); Ras (BD Transduction Laboratories, San Jose, CA; 1:1,000); p53 (DO‐1, BD biosciences); γH2AX(S139) (Upstate, Chicago, IL; 1:1,000); γ‐Tubulin (Sigma, St Louis, MO; 1:5,000).

Techniques: Activation Assay, Recombinant, Western Blot

Journal: Aging Cell

Article Title: Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

doi: 10.1111/acel.12838

Figure Lengend Snippet: hTERT suppresses TGF‐β1‐induced telomere dysfunction and myofibroblast transdifferentiation. (a) Representative micrographs of control treated (top) and TGF‐β1 treated BJ fibroblasts immunostained for α‐SMA (green) and 53BP1 (red). Cell nuclei were counterstained with DAPI (blue). Bar graph: percentage of α‐SMA‐positive BJ fibroblasts that stain positive (dark gray) or negative (light gray) for at least one 53BP1 focus following treatment with TGF‐β1 (10 ng/ml) or control (C) for 48. Error bars: ± SE . * p < 0.05, ( n = 3). Scale bars: 20 μm. (b) Representative micrograph of TGF‐β1 treated BJ fibroblasts immunostained with using antibodies against α‐SMA (white), 53BP1 (green), and a telomeric FISH probe (red). DAPI: (blue). Insets: enlarged area showing indicating nucleus (top) and 53BP1/telomere colocalizations (bottom). Bar graph: percentage of α‐SMA‐positive BJ fibroblasts that were TIF positive (dark gray) or TIF negative (light gray) following treatment with TGF‐β1 (10 ng/ml) or control (C) for 48. Error bars: ± SE . * p < 0.05, ( n = 3). (c) Immunofluorescence staining of early passage BJ fibroblasts (top row), BJ fibroblasts expressing hTERT (+hTERT; center row) and BJ fibroblasts expressing a dominant defective mutant of hTERT (DN‐hTERT; bottom row) treated with TGF‐β1 (10 ng/ml) for indicated times using antibodies against α‐SMA (green) and 53PB1 (red). Nuclear DNA was counterstained with DAPI (blue). Scale bars: 20 µm. (d) Normal BJ fibroblasts (−hTERT; left panel), BJ fibroblasts overexpressing hTERT (+hTERT; center panel), and BJ fibroblasts overexpressing DN‐hTERT (right panel) were treated with TGF‐β1 (10 ng/ml) and solvent control (C) for indicated time points and analyzed by immunoFISH for percentage of TIF‐positive cells (graphs) and by immunoblotting for α‐SMA expression (immunoblots). γ‐tubulin was used as loading control. Error bars: ± SD . * p < 0.05; ns: not significant: p > 0.05 ( n = 3). (e) qRT–PCR analysis of characteristic myofibroblastic genes in the absence of TGB‐β1 treatment (left graph) and following TGB‐β1 treatment for 24 hr (right graph) in normal human BJ fibroblasts (dark bars) and BJ fibroblasts overexpressing hTERT (light bars). Error bars: ± SD , p < 0.05 comparison between ±hTERT for all genes. (f) Fibroblast populated collagen contraction (FPCL) assay. 4 representative wells containing collagen lattices (white disks) contracted by TGF‐β‐treated BJ cells (top row) and BJ cells overexpressing hTERT (bottom row) are shown in left images. Normal BJ fibroblasts (blue line) and hTERT‐expressing BJ fibroblasts (red line) were mixed with rat tail collagen in the presence of TGF‐β1 for 48 hr. Subsequently, lattices were released and allowed to contract freely for indicated times. Graph illustrates percentage decrease in FPCL area, normalized to cell numbers and measured at indicated times following release. Error bars: ± SD . * p < 0.05; ( n = 3)

Article Snippet: The sources and dilutions of antibodies used were as follows: α‐SMA (SIGMA‐Aldrich, 1:1,000), 53BP1 (polyclonal; Novus, Littleton, CO), β‐Tubulin, p53(S15), phospho‐SMAD3 (Cell Signaling, Danvers, MA); p‐ATM(S1981) (Abcam, Cambridge, MA); p21 (Santa Cruz, Dallas, TX), TRF2 (Novus, Littleton, CO; 1:500), p16 (Santa Cruz, CA; 1:200); Nox‐4 (Abcam, Cambridge, MA); macroH2A (kind gift from Dr. Peter Adams), Lamin B1 (Abcam; 1:1,000); Ras (BD Transduction Laboratories, San Jose, CA; 1:1,000); p53 (DO‐1, BD biosciences); γH2AX(S139) (Upstate, Chicago, IL; 1:1,000); γ‐Tubulin (Sigma, St Louis, MO; 1:5,000).

Techniques: Staining, Immunofluorescence, Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR

Journal: Aging Cell

Article Title: Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

doi: 10.1111/acel.12838

Figure Lengend Snippet: TGF‐β1‐induced transdifferentiation is mediated by Smad3/NOX4‐dependent ROS production and telomere dysfunction. (a) Normal BJ fibroblasts (−hTERT; left graph) and BJ fibroblasts overexpressing hTERT (+hTERT; right graph) were either control treated (C) or treated with TGF‐β12 for indicated times. NOX4 mRNA levels relative to controls, which were set to 1, were measured by qRT–PCR. Error bars: ± SD . * p < 0.05, ( n = 3). Immunoblots below graphs illustrate NOX4 and α‐SMA protein levels in extracts from normal BJ fibroblasts (left) and BJ fibroblasts overexpressing hTERT (right) treated with TGF‐β1 for indicated times. (b) Left: Percentage of TIF‐positive BJ fibroblasts that were either control treated (C) or treated with TGF‐β1 for 24 hr in the absence or presence of the Smad3 inhibitor SIS3. Center: quantitation of α‐SMA levels measured by immunoblotting extracts from BJ fibroblasts treated with TGF‐β1 for 24 hr in the absence or presence of the Smad3 inhibitor SIS3. Error bars: ± SD . * p < 0.05, ( n = 3). Representative immunoblot (bottom, γ‐tubulin: loading control) and micrographs (right; α‐SMA: green, 53BP1 red; DAPI: blue) are shown. Scale bars: 500 µm. (c) Same as in B, with the exception that the NOX4 inhibitor VAS2870 (NOX4i) was used instead of SIS3. Error bars: ± SD . * p < 0.05, ( n = 3). (d) Left graph: Percentage of TIF‐positive normal BJ fibroblasts that were either control treated (C) or treated with TGF‐β1 for 24 hr in the absence or presence of the ROS scavenger N ‐acetyl cysteine (NAC). Error bars: ± SD . * p < 0.05, ( n = 3). Center: quantitation of α‐SMA levels measured by immunoblotting extracts from BJ fibroblasts treated with TGF‐β1 for 24 hr in the absence or presence of the NAC. Error bars: ± SD . * p < 0.05, ( n = 3). Representative immunoblot (bottom, γ‐tubulin: loading control) and micrographs (right; αSMA: green, 53BP1 red; DAPI: blue) are shown. Scale bars: 500 µm. (e) Left graph: Percentage of TIF‐positive normal BJ fibroblasts (−hTERT; left graph) and BJ fibroblasts overexpressing hTERT (+hTERT; right graph) that were either control treated (C), treated with increasing concentrations of H 2 O 2 for 24 hr, or with zeocin for 4 hr as indicated. Right graph: quantitation of α‐SMA levels measured by immunoblotting extracts from BJ fibroblasts treated with TGF‐β1 for 24 hr in the absence or presence of H 2 O 2 or zeocin, as indicated. Error bars: ± SD . * p < 0.05, ( n = 3). Representative immunoblot (γ‐tubulin: loading control) and micrographs (right; α‐SMA: green, 53BP1 red; DAPI: blue) are shown. Scale bars: 500 µm

Article Snippet: The sources and dilutions of antibodies used were as follows: α‐SMA (SIGMA‐Aldrich, 1:1,000), 53BP1 (polyclonal; Novus, Littleton, CO), β‐Tubulin, p53(S15), phospho‐SMAD3 (Cell Signaling, Danvers, MA); p‐ATM(S1981) (Abcam, Cambridge, MA); p21 (Santa Cruz, Dallas, TX), TRF2 (Novus, Littleton, CO; 1:500), p16 (Santa Cruz, CA; 1:200); Nox‐4 (Abcam, Cambridge, MA); macroH2A (kind gift from Dr. Peter Adams), Lamin B1 (Abcam; 1:1,000); Ras (BD Transduction Laboratories, San Jose, CA; 1:1,000); p53 (DO‐1, BD biosciences); γH2AX(S139) (Upstate, Chicago, IL; 1:1,000); γ‐Tubulin (Sigma, St Louis, MO; 1:5,000).

Techniques: Quantitative RT-PCR, Western Blot, Quantitation Assay