DOI: 10.5061/dryad.3vc83
title: Data from: Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions

description: Microgravity is a major stress factor that astronauts have to face in
 space. In the past, the effects of microgravity on genomic DNA damage were
 studied, and it seems that the effect on genomic DNA depends on cell types
 and the length of exposure time to microgravity or simulated microgravity
 (SMG). In this study we used mouse embryonic stem (MES) and mouse
 embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA
 lesions. To acquire the insight into potential mechanisms by which cells
 resist and/or adapt to SMG, we also included Rad9-deleted MES and
 Mdc1-deleted MEF cells in addition to wild type cells in this study. We
 observed significant SMG-induced DNA double strand breaks (DSBs) in
 Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type
 cells. A similar pattern of DNA single strand break or modifications was
 also observed in Rad9-/- MES. As the exposure to SMG was prolonged,
 Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced
 DNA lesions. The induced DNA lesions in Rad9-/- MES were due to
 SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF
 cells were only partially adapted to the SMG disturbance. That is, the
 induced DNA lesions were reduced over time, but did not return to the
 control level while ROS returned to a control level. In addition, ROS was
 only partially responsible for the induced DNA lesions in Mdc1-/- MEF
 cells. Taken together, these data suggest that SMG is a weak genomic DNA
 stress and can aggravate genomic instability in cells with DNA damage
 response (DDR) defects.
description: Fig1-A Neutral comet
 assay MES cellFig. 1. Effects of SMG
 on DNA damage and apoptosis in Rad9+/+ and Rad9-/- MES cells (A)
 Evaluation of DNA double strand break by neutral comet assay in Rad9+/+
 and Rad9-/- MES cells cultured under 1G or SMG condition. Time points were
 1, 2, 3, 4 and 5 days. At least 50 cells for each datum were scored for
 comet tail moment.Fig1-BFlow cytometric analysis
 of γ-H2AX formation in Rad9+/+ and Rad9-/- mMES cells cultured under 1G or
 SMG condition for 1 or 5 daysFig1-CEvaluation of DNA damage
 by alkaline comet assay in Rad9+/+ and Rad9-/- MES cells cultured under 1G
 or SMG condition for 1 or 5 days. At least 50 cells of each datum were
 scored for comet tail moment.Fig1-DEvaluation of DNA damage
 by alkaline comet assay in Rad9-/- MES cells with ectopic expression of
 Rad9 (Rad9-/-+Rad9 MES cells) cultured under 1G or SMG condition for 1 or
 5 days. At least 50 cells of each datum were scored for comet tail
 moment.Fig1-EFlow cytometric analysis
 of Rad9+/+ and Rad9-/- MES cells cultured under 1G or SMG condition for 1
 day to assess apoptosis using Annexin V labeling. Experiments were
 performed thrice and representative analyzes are shown (upper). The lower
 part is the quantitative comparison of apoptosis between the 1G Group and
 the SMG Group.Fig2DNA double strand break
 by neutral comet assay in Mdc1+/+ and Mdc1-/- MEF cells cultured under 1G
 or SMG condition for 1 or 5 days. At least 50 cells of each datum were
 scored for comet tail moment.Fig3
 a,b,c(A) Flow cytometric
 analysis of ROS activity in Rad9+/+ and Rad9-/- MES cells exposed to 1G or
 SMG condition for 1 or 5days. (B) Flow cytometric analysis of ROS activity
 in Rad9-/-+Rad9 MES cells exposed to 1G or SMG condition for 1 or 5days.
 (C) N-acetylcysteine inhibited SMG-induced increase of ROS formation in
 Rad9-/- MES cells. Rad9-/- MES cells were mock-treated or treated with
 0.05, 0.1 or 0.5 mM N-acetylcysteine under SMG for 1
 day.Fig3
 A,B,C.xlsFig3 d
 e(D)
 Evaluation of DNA damage by alkaline comet assay in Rad9-/- MES cells
 mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG
 condition for 1 day. (E) Evaluation of DNA damage by neutral comet assay
 in Rad9-/- MES cells mock-treated or treated with 0.5 mM N-acetylcysteine
 under 1G of SMG condition for 1 day.Fig3
 D,E.xlsFig4(A) Flow cytometric
 analysis of ROS activity in Mdc1+/+ and Mdc1-/- MEF cells exposed to 1G or
 SMG condition for 1 or 5days.(B) N-acetylcysteine inhibited SMG-induced
 increase of ROS formation in Mdc1-/- MEF cells. Mdc1-/- MEF cells were
 mock-treated or treated with 0.05, 0.1 or 0.5 mM N-acetylcysteine under
 SMG for 1 day. (C) Evaluation of DNA damage by neutral comet assay in
 Mdc1-/- MEF cells mock-treated or treated with 0.5 mM N-acetylcysteine
 under 1G of SMG condition for 1 day.Fig5(A) Quantitative
 real-time PCR analysis of Nox2 mRNA expression in Rad9+/+ and Rad9-/- MES
 cells exposed to 1G or SMG condition for 1 or 5days. The expression levels
 of Nox2 were normalized to the endogenous control GAPDH expression. (B)
 Quantitative real-time PCR analysis of Nox4 mRNA expression in Rad9+/+ and
 Rad9-/- MES cells exposed to 1G or SMG condition for 1 or 5days. The
 expression levels of Nox4 were normalized to the endogenous control GAPDH
 expression. (D) Quantitative comparison of Nox2 expression. Data were
 derived from three independent experiments . The expression levels of Nox2
 protein were normalized to the endogenous control GAPDH protein
 expression.Fig6-AHistograms of superoxide
 dismutase enzyme activityFig6.A.xlsFig6-BHistograms of catalase enzyme activityFIG6.B.xlsFig6-CHistogram of Glutahione peroxidaseFig6.C.xlsFig7Quantitative comparison of Nox2 expression. Data were derived from three independent experiments The expression levels of Nox2 were normalized to the endogenous control GAPDH expressionFig8A(A) The initial MES cells seeding number was 3×104. The doubling generation curve was generated by dividing the cell number with 104 and then transferring the quotient to the logarithm to the base2.Fig8-A.xlsFig8BThe initial MEF cells seeding number was 105. The doubling generation curve was generated by dividing the cell number with 104 and then transferring the quotient to the logarithm to the base2.FIG8-B.xls