Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: RNF8 promotes ALT-EJ. ( A ) RNA analysis of an Rnf8-/- mESC line generated using Cas9 and sgRNAs targeting Rnf8 . Shown are RT-PCR amplification products for a region of the RNF8 mRNA from exons 1–6 (Ex1–Ex6), and Actin control, for RNA from WT and Rnf8-/- mESCs, treated with and without reverse transcriptase (RT). ( B ) The Rnf8-/- mESC line shows a defect in 53BP1 foci formation. Shown are representative fluorescent images of WT and Rnf8-/- mESCs treated with 6 Gy and recovered for 4 h, stained with DAPI and antibodies for γH2AX and 53BP1. Scale bar = 20 μm. Also shown are violin plots depicting the number of γH2AX foci per nucleus (left) and 53BP1 foci per nucleus (right) in WT and Rnf8-/- mESC. N = 120. (*) P < 0.001 for WT versus Rnf8-/- measuring γH2AX or 53BP1 foci using the Mann–Whitney test. ( C ) RNF8 promotes ALT-EJ, and inhibits EJ without indels (i.e. C-NHEJ). Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, EJ6-GFP+TREX2, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Rnf8-/- mESCs and Rnf8-/- mESCs transfected with a 3×Flag-tagged RNF8 expression vector (3×F-RNF8). WT EJ7-GFP and EJ6-GFP+TREX2 values are from Figure and shown here for comparison. As with all mESC experiments in this study, the reporters are integrated into chromosome 17 at the Pim1 locus. Error bars represent SD. N ≥ 6. (*) P ≤ 0.038 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for Flag, and Actin control, in Rnf8-/- mESCs transfected with and without 3×F-RNF8. (*) Non-specific bands. ( D ) RNF8 promotes ALT-EJ in mESCs, as measured by the EJ2-GFP reporter. Shown are GFP+ frequencies for EJ2-GFP in WT and Rnf8-/- mESCs with and without complementation vector. N = 12. Error bars represent SD. (*) P ≤ 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) RNF8 promotes ALT-EJ in HEK293 cells, as measured by the EJ2-GFP reporter. Two independent RNF8 knockout ( RNF8-KO ) cell lines ( RNF8-KO clones A and B) were generated in an HEK293 cell line with the EJ2-GFP reporter. Shown are GFP+ frequencies for the EJ2-GFP assay for these two cell lines with and without an RNF8 expression vector, as well as for the HEK293 WT cell line. N = 6. Error bars represent SD. (*) P ≤ 0.001766 for unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot analysis for RNF8, and Actin control, for the two RNF8-KO HEK293 cell lines transfected with RNF8, or only control EV, along with HEK293 WT cells transfected with EV.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, MANN-WHITNEY, Transfection, Expressing, Plasmid Preparation, Western Blot, Knock-Out, Clone Assay

Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: 53BP1 does not have a substantial effect on either EJ without indels or ALT-EJ, whereas POLQ promotes ALT-EJ independently of KU. ( A ) Influence of 53BP1 on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, 53bp1-/- mESCs and 53bp1-/- mESCs transfected with an expression vector for human 53BP1. Also shown are GFP+ frequencies for 4-μHOM Embed in 53bp1-/-Rnf8-/- mESCs transfected with and without 53BP1 and RNF8 (3×F-RNF8) expression vectors, and compared to 53bp1-/- mESCs. WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P ≤ 0.049 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for 53BP1 and Actin control in WT and 53bp1-/- mESCs transfected with and without 53BP1. (*) represent non-specific bands. ( B ) Influence of POLQ on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Polq-/- mESCs and Polq-/- mESCs transfected with human Flag-tagged POLQ (POLQ). WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( C ) Immunoblot analysis of KU70, Flag-POLQ and Actin control. A double mutant Ku70-/-Polq-/- mESC line was generated to examine the influence of POLQ and KU70 on EJ independently of the other factor. Shown are KU70 immunoblot signals for WT, and Ku70-/-Polq-/- mESCs transfected with and without KU70. Also shown are Flag and Actin control immunoblot signals for Ku70-/-Polq-/- mESCs transfected with and without Flag-tagged POLQ. (*) indicates a non-specific band. ( D ) POLQ and KU70 independently mediate distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, and Ku70-/-Polq-/- mESCs without complementation vector, or with expression vectors for KU70 or POLQ. WT values for EJ7-GFP, and 4-μHOM are from Figures and , respectively, and shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) Shown is a model for the influence of RNF8 on chromosomal break repair outcomes.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Mutagenesis, Generated

Journal: Nucleic Acids Research

Article Title: Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

doi: 10.1093/nar/gkr149

Figure Lengend Snippet: A novel human lung three-dimensional culture model for the study of differentiated cells response to DSBs. ( A ) Human bronchial epithelial cells (HBECs) form well-organized, growth-arrested structures when cultured in extracellular matrix and on top of IMR90 human lung fibroblasts. Representative optical sections and deconvoluted confocal microscopic images of 3D structures are shown. HBECs stably expressing EGFP-53BP1 grown in Matrigel for 6 days were immunostained with antibodies to α-actin, E-cadherin, SP-A, pH3, Ki-67 and 53BP1. The images were acquired using confocal microscopy and deconvoluted using Imaris software. ( B and C ) HBECs grown in 3D culture have fewer S-phase and more G1-phase cells than in 2D culture. Flow cytometric profiles of the cell cycle distribution of (B) 3D and (C) 2D cultures at the time of irradiation. HBECs in 2D and 3D cultures were pulse labeled with BrdU and then subjected to immunostaining and ‘Flow cytometry’ as described in ‘Materials and Methods’ section. Data are representative of at least three independent experiments. ( D ) Number of cells in 3D structures did not increase between 6 and 10 days in Matrigel culture. Images of 3D structures were acquired at Days 6, 7, 8 and 10 using confocal microscopy. The number of cells per 3D structure was counted using Imaris software. Cells in more than 100 3D structures were counted each day. ( E ) Cells in 3D structures have fewer spontaneous EGFP-53BP1 foci than cells in 2D culture. HBECs stably expressing EGFP-53BP1 grown in 2D and 3D cultures were immunostained with antibodies to 53BP1. The images were acquired using confocal microscopy and deconvoluted using Imaris software. More than 1000 2D cells and 100 3D structures were evaluated; error bars represent standard deviations calculated from three independent experiments.

Article Snippet: To generate the HBEC-EGFP 53BP1 stable line, HBECs were transfected with linearized EGFP-53BP1 mammalian expression plasmid (pcDNA, Invitrogen) using Nucleofector (Amaxa).

Techniques: Cell Culture, Stable Transfection, Expressing, Confocal Microscopy, Software, Irradiation, Labeling, Immunostaining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

doi: 10.1093/nar/gkr149

Figure Lengend Snippet: Number of DNA DSBs formed in 3D structures is dose- and LET-dependent. ( A ) Representative deconvoluted images of 3D structures showing co-localization of EGFP-53BP1 with γH2AX and DNA-PKcs (pT2609) after exposure of cells to graded doses of γ-rays and Fe particles. HBECs stably expressing EGFP-53BP1, grown in Matrigel for 6 days, were irradiated with indicated doses of γ-rays and Fe particles and were fixed at 30 min after irradiation. Subsequently, cells were immunostained with anti-γH2AX and DNA-PKcs (pT2609) antibodies and images were recorded using confocal microscopy. ( B ) Graph showing the number of EGFP-53BP1 foci formed at 30 min after exposure of 2D and 3D cultures to 10–100 cGy of γ-irradiation. ( C ) Graph showing the number of EGFP-53BP1 foci formed at 30 min after exposure of 2D and 3D cultures to 10–100 cGy of Fe particles irradiation. HBECs stably expressing EGFP-53BP1 grown in 2D and 3D cultures were irradiated with indicated doses of γ-rays and Fe particles and were fixed at 30 min after irradiation. The images were acquired using confocal microscopy and the foci numbers were counted using spot-detection function of Imaris software. The EGFP-53BP1 foci numbers in 200–400 cells from 2D culture and 15–18 3D structures were counted for each dose. The error bars represent standard deviations calculated from three independent experiments.

Article Snippet: To generate the HBEC-EGFP 53BP1 stable line, HBECs were transfected with linearized EGFP-53BP1 mammalian expression plasmid (pcDNA, Invitrogen) using Nucleofector (Amaxa).

Techniques: Stable Transfection, Expressing, Irradiation, Confocal Microscopy, Software

Journal: Nucleic Acids Research

Article Title: Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

doi: 10.1093/nar/gkr149

Figure Lengend Snippet: Ionizing radiation-induced DSBs are repaired with slower kinetics in 3D structures than in cells in 2D culture. ( A ) Representative deconvoluted images of 3D structures showing EGFP-53BP1 foci at indicated times after 1 Gy of γ-rays and Fe particles irradiation. HBECs stably expressing EGFP-53BP1 grown in Matrigel for 6 days were irradiated with 1 Gy of γ-rays and Fe particles and were fixed at indicated times after irradiation. Subsequently, images were recorded using confocal microscopy. ( B ) EGFP-53BP1 foci dissolution kinetics are slower in 3D than 2D cultures after 1 Gy of γ-rays or ( C ) Fe particles irradiation. HBECs stably expressing EGFP-53BP1 grown either in 2D or Matrigel, irradiated with 1 Gy of γ-rays and Fe particles and fixed at indicated times. Subsequently, images were acquired using confocal microscopy and the EGFP-53BP1 foci numbers were counted using spot-detection function of Imaris software. The number of EGFP-53BP1 foci in the mock-irradiated cells was subtracted and data were plotted by taking the number of foci at the 30 min time point as 100%. The EGFP-53BP1 foci in 150–200 cells from 2D culture and 15–18 3D structures were counted for each time point. The error bars represent standard deviations calculated from three independent experiments.

Article Snippet: To generate the HBEC-EGFP 53BP1 stable line, HBECs were transfected with linearized EGFP-53BP1 mammalian expression plasmid (pcDNA, Invitrogen) using Nucleofector (Amaxa).

Techniques: Irradiation, Stable Transfection, Expressing, Confocal Microscopy, Software

Journal: Nucleic Acids Research

Article Title: Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

doi: 10.1093/nar/gkr149

Figure Lengend Snippet: Non-linear regression-dynamic fitting of EGFP-53BP1 foci dissolution kinetics

Article Snippet: To generate the HBEC-EGFP 53BP1 stable line, HBECs were transfected with linearized EGFP-53BP1 mammalian expression plasmid (pcDNA, Invitrogen) using Nucleofector (Amaxa).

Techniques:

Journal: Nucleic Acids Research

Article Title: Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

doi: 10.1093/nar/gkr149

Figure Lengend Snippet: Iron particles-induced complex DSBs persist in 2D and 3D cultures. ( A ) Representative deconvoluted images showing colocalization of persistent EGFP-53BP1 foci with phosphorylated H2AX (γH2AX) and Chk2 (pChk2) in 2D and ( B ) 3D structures. HBECs stably expressing EGFP-53BP1 grown either in 2D or Matrigel, irradiated with indicated doses of Fe particles and were fixed after 5 days. Cells were then immunostained with anti-γH2AX and pChk2 (pT68) antibodies. Images were acquired using confocal microscopy. ( C ) A majority of Fe particles-induced complex DSBs persist in 3D structures. HBECs stably expressing EGFP-53BP1 grown either in 2D or Matrigel, irradiated with indicated doses of Fe particles and fixed after 5 days. Images were acquired using confocal microscopy and the EGFP-53BP1 foci numbers were counted using spot-detection function of the Imaris software. For each dose, the number of EGFP-53BP1 foci in the mock-irradiated cells was subtracted. The EGFP-53BP1 foci in 150–200 cells from 2D culture and 15–18 3D structures were counted for each dose. The error bars represent standard deviations calculated from three independent experiments. ( D ) Cells containing irreparable complex DNA DSBs generated by Fe particles in 3D structures are differentiated. Representative deconvoluted images showing localization of EGFP-53BP1 foci in the differentiated cells (Ki-67 negative) of 3D structures. HBECs stably expressing EGFP-53BP1 were grown in Matrigel for 6 days, exposed to 1 Gy of Fe particles and were fixed at 5 days after irradiation. Subsequently, cells were immunostained with anti-Ki-67 antibodies and the images were acquired using confocal microscopy and deconvoluted using Imaris software.

Article Snippet: To generate the HBEC-EGFP 53BP1 stable line, HBECs were transfected with linearized EGFP-53BP1 mammalian expression plasmid (pcDNA, Invitrogen) using Nucleofector (Amaxa).

Techniques: Stable Transfection, Expressing, Irradiation, Confocal Microscopy, Software, Generated

Journal: Autophagy

Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites

doi: 10.4161/auto.23689

Figure Lengend Snippet: Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites (A) and cannot complement yeast Atg8 (B). (A) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. (B) Scatg8Δ and wt strains were transformed with empty pFL61 plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.

Article Snippet: The fragments were cloned into the yeast expression vector pFL61 53 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method.

Techniques: Infection, Expressing, Staining, Labeling, Transformation Assay, Plasmid Preparation, Positive Control, Western Blot, Functional Assay