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: The number of cells per 3D structure was counted using Imaris software.

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: The number of cells per 3D structure was counted using Imaris software.

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: The number of cells per 3D structure was counted using Imaris software.

Techniques: Irradiation, Stable Transfection, Expressing, Confocal Microscopy, Dissolution, 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: 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: The number of cells per 3D structure was counted using Imaris software.

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