Journal: PLoS Pathogens

Article Title: Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining

doi: 10.1371/journal.ppat.1009812

Figure Lengend Snippet: A, Distributions of mapped RNA-seq reads for H2ax, Fen1, Cdk1, Plk1, Polθ, and Mdc1 in tumor (Sample #4) and tumor-free (sample #11) ear tissues ( see ) to the mouse genome were visualized by IGV. B, Quantitative RNA expression for H2ax, Fen1, Cdk1, Plk1, Polθ, and Mdc1 from ear tissues by RT-qPCR. Total RNA extracted from MmuPV1 tumor (sample #4) and tumor-free (sample #11) ear tissues from animal #2 ( See ). The RNA expression levels of the indicated genes were determined by RT-qPCR using mouse gene-specific TaqMan probes. C, Phosphor-H2AX (S139) (γH2AX), FEN1, CtIP, and phosphor-CtIP (S327) in tumor samples #1 and #4 and tumor-free samples #10 and #12) ( see ) were determined by Western blot using the indicated antibodies. Tubulin served as a sample loading control.

Article Snippet: RT-qPCR was carried out using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, #4369016) with gene-specific probes of Cdk1(#Mm00772472_m1), Plk1(#Mm00440924_m1), Fen1 (#Mm01700195_m1), Polθ (#Mm01170059_m1), Mdc1(#Mm01273851_g1), or H2ax (Mm00515990_s1).

Techniques: RNA Sequencing, RNA Expression, Quantitative RT-PCR, Western Blot, Control

Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 6. Deregulation of TRIP12 and UBR5 Alters the Dynamics of DNA Repair (A) U-2-OS cells were transfected with the indi- cated siRNA for 72 hr and irradiated. WCE were analyzed by immunoblotting. (B) U-2-OS cells were irradiated (2 Gy), and 1 hr later, they were immunostained with an antibody to RPA (the FITC channel); nuclear DNA was coun- terstained by DAPI. Cell-cycle distribution was determined for each individual cell by quantifying the total DAPI and chromatin-bound RPA intensity per nucleus. (C) U-2-OS cells were transfected with the indi- cated siRNAs, irradiated, immunostained with antibodies to RPA and RAD51, sorted according to cell cycle as in (B), and subjected to an automated analysis of RAD51 focus formation. At least 500 cells were analyzed for each condition; represen- tative images are shown. (D) U-2-OS cells were transfected with siRNAs for 72 hr and irradiated. Where indicated, cells were treated for 2 hr with ATM or DNA-PK inhibitors prior to irradiation. WCE were analyzed by immuno- blotting (left). In parallel, cells were treated with the indicated siRNAs and inhibitors as indicated, irra- diated (2 Gy), and after 4 hr, were subjected to an automated single cell analysis for the number of MDC1 nuclear foci (right). (E) U-2-OS cells were treated with the indicated siRNAs and inhibitors, irradiated, and analyzed as in (D). Scale bar, 10 mm. See also Figure S7.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Irradiation, Western Blot, Single-cell Analysis

Journal: Developmental Cell

Article Title: SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice

doi: 10.1016/j.devcel.2018.10.004

Figure Lengend Snippet:

Article Snippet: sheep MDC1 , Serotec , AHP799; RRID: AB_323725.

Techniques: Software

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

Article Title: BRCT-domain protein BRIT1 influences class switch recombination

doi: 10.1073/pnas.1708211114

Figure Lengend Snippet: BRIT1 and MDC1 could both contribute to the ATM–γ-H2AX axis of DSB resolution during CSR. (A) Representative flow cytometry of CSR to IgG1 in splenic B cells derived from Mb1-Cre control, BRIT1 KO, and AID KO mice treated with ATMi or with DMSO vehicle control. (B) Quantification of CSR to IgG1 (n = 3; **P < 0.005, *P < 0.05). (C) Immunoblot analysis of whole-cell lysates from control, BRIT1 KO, or AID KO cells treated with ATMi or DMSO. (D) Immunoblot analysis of whole-cell lysates from control or BRIT1 KO cells transduced with two different MDC1 shRNAs (sh1 and sh2) or scrambled control. (E) Representative flow cytometry of CSR to IgG1 in splenic B cells of indicated genotypes expressing MDC1 knockdown shRNAs sh1 or sh2. (F) Quantification of CSR to IgG1 (n = 3; **P < 0.005, *P < 0.05).

Article Snippet: The shRNAs (sh3 and sh4) against mouse MDC1 were obtained from Origene (TG517490).

Techniques: Flow Cytometry, Derivative Assay, Western Blot, Transduction, Expressing

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

Article Title: BRCT-domain protein BRIT1 influences class switch recombination

doi: 10.1073/pnas.1708211114

Figure Lengend Snippet: Depletion of MDC1 exacerbates CSR defect in BRIT1 KO B cells. (A) Strategy to deplete MDC1 in ex vivo stimulated splenic B cells. B cells were harvested from control and BRIT1 KO mice, activated with LPS + IL-4, and retrovirally infected with shRNA and a GFP marker (sh3 and sh4). WB, Western blot. (B) Representative flow cytometry of CSR to IgG1 from Mb1-Cre control and BRIT1 KO mice with MDC1 knockdown constructs sh3 and sh4. (C) Quantification of CSR to IgG1 (n = 3; **P < 0.005, *P < 0.05). (D) Immunoblot analysis of BRIT1, MDC1, and AID upon MDC1 depletion.

Article Snippet: The shRNAs (sh3 and sh4) against mouse MDC1 were obtained from Origene (TG517490).

Techniques: Ex Vivo, Infection, shRNA, Marker, Western Blot, Flow Cytometry, Construct

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

Article Title: BRCT-domain protein BRIT1 influences class switch recombination

doi: 10.1073/pnas.1708211114

Figure Lengend Snippet: shRNA sequences against mouse MDC1 and scramble control

Article Snippet: The shRNAs (sh3 and sh4) against mouse MDC1 were obtained from Origene (TG517490).

Techniques: shRNA, Plasmid Preparation