DataSheet2_A new type of simulated partial gravity apparatus for rats based on a pully-spring system.pdf

The return to the Moon and the landing on Mars has emphasized the need for greater attention to the effects of partial gravity on human health. Here, we sought to devise a new type of simulated partial gravity apparatus that could more efficiently and accurately provide a partial gravity environment for rat hindlimbs. The new apparatus uses a pulley system and tail suspension to create the simulated partial gravity of the rat’s hind limbs by varying the weight in a balance container attached to the pulley system. An experiment was designed to verify the reliability and stability of the new apparatus. In this experiment, 25 seven-week-old male Wistar Hannover rats were randomly divided into five groups (n = 5 per group): hindlimb full weight-bearing control (1G), sham (1G), and the simulated gravity groups including Mars (3/8G), Moon (1/6G), and interplanetary space (microgravity: µG). The levels of partial gravity experienced by rat hindlimbs in the Mars and Moon groups were provided by a novel simulated partial gravity device. Changes in bone parameters [overall bone mineral density (BMD), trabecular BMD, cortical BMD, cortical bone thickness, minimum moment of area (MMA), and polar moment of area (PMA)] were evaluated using computed tomography in all rats at the proximal, middle, and distal regions of femur and tibia. Reduced gravity led to decreases in bone parameters (overall BMD, trabecular BMD, cortical BMD, MMA, and PMA) in the simulated gravity groups, mainly in distal femur and proximal tibia. The proximal tibia, MMA, and PMA findings indicated greater weakness in the µG group than in the Mars group. The sham group design also excluded the decrease in lower limb bone parameters caused by the suspension attachment of the rat’s tail. The new simulated partial gravity apparatus can provide a continuous and stable level of partial gravity. It offers a reliable and valuable model for studying the effects of extraterrestrial gravity environments on humans.</p

Additional file 1: Figure S1. of Combining carbon ion irradiation and non-homologous end-joining repair inhibitor NU7026 efficiently kills cancer cells

Survival curves after radiation exposure with NU7026 or B02 treatment in human glioblastoma cells. A172 cells (A and B), U251MG cells (C and D). NU7026 treatment (A and C), B02 treatment (B and D). The presented results are the mean and SD of three independent experiments. Data were statistically evaluated with student’s t test with comparisons between inhibitor alone and other treated groups (* p < 0.05; ** p < 0.01; *** p < 0.001). (PDF 122 kb

Additional file 2: Figure S2. of Combining carbon ion irradiation and non-homologous end-joining repair inhibitor NU7026 efficiently kills cancer cells

Radiosensitization with NU7026 or B02 treatment in human glioblastoma cells. Survival curves (A and B), Sensitization ratio values (C and D). A172 cells (A and C), U251MG cells (B and D) after exposure to C ion irradiation. The presented results are the mean and SD of three independent experiments. Data were statistically evaluated with the student’s t test with comparisons between irradiation alone and other treated groups (* p < 0.05; ** p < 0.01). (PDF 119 kb

Effects of KU55933 (KU) and NU7026 (NU) on US- or IR-induced γH2AX signaling.

<p>(A) Immunoblots of U937 cell extracts 30 min after US or IR and effects of KU and NU on ATM pS1981 and DNA-PKcs pS2056/pT2609. (B) Greater suppression of US-induced γH2AX by NU than KU up to 6 h after US in U937 cells in WB analysis. (C) Effects of KU and/or NU: a greater suppression of US-induced γH2AX+ cells by NU than KU, and abrogation by KU-plus-NU in FCM analysis. (<i>n</i> = 3, mean ± s.d.). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Cells were treated with KU and/or NU (10 µmol/L) for 1 h before and after exposure to 0.3 W/cm² US (i) or 10 Gy IR (ii). (D) DNA-PK preceded ATM for γH2AX induction by US: a preferential role of DNA-PK in γH2AX induction was determined by immunostaining. Cells were treated as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029012#pone-0029012-g004" target="_blank">Fig. 4C</a>. ATM pS1981 (AT), DNA-PKcs pS2056 (PK), and γH2AX (H2) positive/negative cells were counted at least 100 cells in each experiment. The data shows the averages from 2 independent experiments.</p

Induction and repair of DSBs and γH2AX foci after US or IR.

<p>(A) SYBR green-stained neutral comet tails immediately after exposure of U937 (<i>U</i>), Jurkat (<i>J</i>), Molt-4 (<i>M</i>) and HL-60 (<i>H</i>) cells to US (0.3 or 0.4 W/cm²) or IR (10 Gy). (B) Relative tail moments (<i>n</i> = 100 cells, means ± SD), normalized to the respective untreated controls ( = 1.0). (C) Heterogeneous distribution to >3.0, 1.1∼3.0 and 1 ( = control level) mean relative tail moments after US, but a uniform distribution to 1.1–3 relative tail moment after 10 Gy in ∼90% U937 cells (<i>n</i> = 100 cells). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029012#pone.0029012.s001" target="_blank">Fig. S1</a> for other cell lines. (D) Green-fluorescentγH2AX images in U937, Jurkat, Molt-4 and HL-60 cells 30 min after 0.3 W/cm² US (control cell images were not shown due to no γH2AX+ cells). (E) Induction of γH2AX+ cells as a function of US intensity beyond a threshold of 0.1–0.2 W/cm² (<i>n</i> = 3, mean ± s.d.). (F) γH2AX+ cell images and (G) FCM histograms of γH2AX+ U937 cells 30 min after 0.3 W/cm² and 10 Gy IR. Black, green, and red profiles are for control, US, and IR, with MFIs of γH2AX+ cells (5–100 γH2AX log)). (H) Induction/decline of γH2AX+ U937 cells (FCM) with time after 0.3, 0.4 W/cm² (i) and 10 Gy IR (ii). (I) Reduction in tail moments during 3 h post- 0.3 W/cm² US (i) or 10 Gy IR (ii). zVAD-fmk at 50 µmol/L was used to eliminate apoptotic DSBs.</p

Linking mechanical US effect withinduction of γH2AX+ cells.

<p>(A) EPR detection of US- or IR-induced extra- and intracellular levels of OH• as DMPO-OH adducts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029012#s3" target="_blank">Methods</a>); Increase of OH• levels in DMPO solution (10 mmol/L) as a function of insonation time at 0.3 (<i>left</i>) and 0.4 (<i>middle</i>) W/cm² US, or at IR dose of 5–20 Gy (<i>right, closed circle</i>). Induction of DMPO-OH adducts by 0.3 or 0.4 W/cm² were reduced partially by 50 mmol/L DMSO (<i>upward triangle</i>) or 5 mmol/L NAC (<i>downward triangle</i>), and nullified by a 10-fold higher concentrations: 50 mmol/L NAC or 500 mmol/L DMSO (<i>closed diamond</i> for both). The insets show amplitudes of EPR signals of DMPO-OH. (B) FCM-based HPF assay for intracellular OH• levels immediately after 0.3 W/cm² US in U937 cells. The histogram shift toward high-HPF fluorescence by OH• oxidation was small, and thus, an increase in mean fluorescence intensity (MFI) was 1.57±0.07 fold the control (<i>n</i> = 3, mean ± s.d.), with partial protection by pretreatment with 5 mmol/L NAC for 3 h before sonication. (C) No protective effects of 5 mmol/L DMSO or 5 mmol/L NAC (scavengers) added to cultures immediately before 0.3 and 0.4 W/cm² US, or other 3 h-pretreatment with 5 mmol/L NAC (NAC-pre) against the induction of γH2AX+ U937 cells 30 min post-US. (<i>n</i> = 3, mean ± s.d. <i>ns</i> means <i>not significant</i>). (D–F) Saturated N₂O gas caused the abrogation of US-induced events as follows: (D) The US exposure-time dependent induction of OH• in 10 mmol/L DMPO solution at 0.3 and 0.4 W/cm² (<i>n</i> = 3, mean ± s.d.). : (E) The induction of γH2AX+ U937 cells 30 min after 0.3 and 0.4 W/cm² (<i>n</i> = 3, mean ± s.d.). : (F) 20% non-viable cells (trypan blue dye exclusion test) and 30% loss in cell counts relative to control U937 cells 6 h after 0.3 or 0.4 W/cm² US (<i>n</i> = 3, mean ± s.d.).</p

Differential ATM and DNA-PK signaling to γH2AX in response to US or IR.

<p>(A) Colocalization of distinct NBS1 pS343 foci to distinct γH2AX foci; (B) Colocalization of ATM pS1981 foci to γH2AX foci; (C) DNA-PKcs pT2609 foci largely independent of γH2AX foci. (D) US- and IR-induced DNA-PKcs pS2056 and γH2AX foci. US induced peri-nuclear high-fluorescent DNA-PKcs pS2056 foci (<i>right</i>) or were pan-nuclear with discrete foci <i>(left)</i>, whereas both foci after IR were distinct and colocalized. Fluorescent images were acquired 30 min after 0.3 W/cm² US and 3 Gy IR in U937 cells.</p

A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.

<p>A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.</p

Rad51 foci formation in glioblastoma A172 cells after ACNU and TMZ treatments.

<p><b>a</b>, typical photo. <b>b</b>, open columns, negative control siRNA; closed columns, <i>FANCD1/BRCA2</i> siRNA. Columns show the mean of two independent experiments; bars indicate the SD.</p

Sensitivity to TMZ.

<p><b>a</b>, <i>FANCwt</i> cells (open circles), <i>FANCA^−/−C^−/−</i> cells (closed triangles), <i>FANCC^−/−</i> cells (closed squares), and <i>FANCA^−/−</i> cells (closed circles); <b>b</b>, <i>FANCwt</i> cells (open circles) and <i>FANCD2^−/−</i> cells (closed circles); <b>c</b>, <i>FANCGwt</i> cells (open circles), <i>FANCGrev</i> cells (closed triangles), and <i>FANCG^−/−</i> cells (closed circles); <b>d</b>, <i>FANCD1wt</i> cells (open circles), <i>FANCD1rev</i> cells (closed triangles), and <i>FANCD1mt</i> cells (closed circles). Each point represents the mean of three independent experiments; bars indicate the SD.</p