Additional file 1: of Non-coplanar volumetric-modulated arc therapy (VMAT) for craniopharyngiomas reduces radiation doses to the bilateral hippocampus: a planning study comparing dynamic conformal arc therapy, coplanar VMAT, and non-coplanar VMAT

The dataset supporting our findings. (XLSX 26 kb

Imaging of HIF-1-active regions in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. Magnified head images are shown in the lower left panels. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere. (B) The relative fluorescence intensity of the ischemic hemisphere to the non-ischemic hemisphere. Fluorescence intensities were measured at the indicated times after POH-N administration. *<i>P</i><0.05, n = 3. (C) <i>Ex vivo</i> imaging of the coronal brain sections after POH-N injection. (D) Relative fluorescence of the ischemic hemisphere compared with the non-ischemic hemisphere at 6 h after probe administration (n = 3/group: *<i>P</i><0.05). Relative fluorescence values were calculated using ROIs mirrored along the midline of the cerebral hemispheres. (E) <i>In vivo</i> fluorescence images visualized without preparation of a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration. Anesthetized C57BL/6J mice were shaved and depilated top of the head 24 h before experimentation. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere.</p

No clear visualization of HIF-1-active regions in the permanent brain ischemia model or with delayed injection of POH-N in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. POH-N was injected intravenously at 60 min after permanent MCA occlusion. (B) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h following POH-N administration at 24 h after reperfusion. Magnified head images are shown in the lower left panels.</p

Stabilization of POH-N under hypoxic conditions.

<p>SH-SY5Y neuroblastoma cells cultured under normoxic (N) or hypoxic (H) conditions were treated with POH probe. (A) HIF-1α protein levels were analyzed by western blotting (a representative blot is shown). (B) The fluorescence intensity of POH probe in cells was measured. (C) Representative fluorescence images are shown. *<i>P</i><0.02 (vs. normoxic condition).</p

Replication-blocking agents induce comparable numbers of chromosome breaks in both DSB-repair-proficient and -deficient chicken DT40 and human Nalm-6 cell lines.

<p>(A–D) Frequency of chromosomal aberrations (CAs) in <i>wild-type</i> and <i>RAD54^−/−/KU70^−/−</i> DT40 cells before (0) and after treatment with (A) γ-irradiation, (B) 5-FU, (C) HU, and (D) aphidicolin (APH). Cells were analyzed at 3 h after irradiation (A). Cells were incubated with 5-FU or HU for 24 h, or with aphidicolin for 48 h at the indicated concentrations (B–D). In each case, cells were treated with colcemid for the last 3 h. More than 100 cells were analyzed in each case. (E–H) Frequency of chromosomal aberrations (CAs) in <i>wild-type</i> and <i>RAD54^−/−/LIG4^−/−</i> Nalm-6 cells before (0) and after treatment with (E) γ-irradiation, (F) 5-FU, and (G) HU. Cells were incubated with 5-FU or HU for 48 h at the indicated concentrations (F, G). (H) The number of induced CAs was calculated by subtracting the number of non-treated cells from the number of cells treated with γ-rays or chemicals. More than 50 cells were analyzed at 8 h after irradiation at 0.1 Gy. More than 100 cells were analyzed for 5-FU and HU. Error bars show standard error, based on the Poisson distribution of spontaneous chromosomal aberrations observed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone.0060043-Sonoda2" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone.0060043-Kikuchi1" target="_blank">[52]</a>.</p

Immunohistochemical detection of HIF-1-active cells and POH-N probe.

<p>(A) Immunohistochemical analysis of HIF-1α, POH-N (ODD) and HaloTag (green), with or without DAPI nuclear staining (blue), at 1 day after probe administration. Panels at the bottom show magnified images. (B) Similar distributions of HIF-1α, HaloTag, and HSP70 in pyramidal neurons of the cortical layer bordering the infarct. Scale bars, 50 μm.</p

Determination of Gβγ interacting domain of the AGS8.

<p>(A) Schematic diagram of rat AGS8 and the AGS8 domains synthesized as GST-fusion proteins. Each GST protein fused with the following segment of rat AGS8 (ABB82299) respectively. AGS8-N: M¹- P³⁷⁰, AGS8-254: A²⁵⁴– R⁵⁵³, AGS8-534: S⁵³⁴– S⁸³³, AGS8-814: S⁸¹⁴– R¹¹¹³, AGS8-1094: H¹⁰⁹⁴– D³²⁸⁰, AGS8-C: A¹³⁵⁹– W¹⁷³⁰. (B) GST-pulldown assay of AGS8 domains with Gβ₁γ₂. AGS8 domains synthesized as GST-fusion proteins (300 nM) were incubated with recombinant human Gβ₁γ₂ (30 nM) in a total volume of 300 µl at 4°C. Proteins were then adsorbed to a glutathione matrix and retained G-protein subunits identified by immunoblotting following gel electrophoresis. The representative of 5 independent experiments with similar results. (C) Bioactivity of AGS8 domains on G-protein activation in cell. The yeast strain expressing human Gαs was transformed with AGS8 domains described in (A) into the pYES2-containing GAL1 promoter. The yeast strain was modified to grow without histidine on activation of G-protein. Induction(+): induction of translation of AGS8 domains by galactose. The representative of 4 independent experiments with similar results. (D) GST-pull down assay of AGS8-C segments with recombinant Gβγ. (<i>upper panel</i>) Schematic diagram of AGS8-C and the segments synthesized as GST-fusion proteins. Each GST protein fused with the following segment of rat AGS8 (ABB82299) respectively. AGS8-C1: A¹³⁵⁹– H¹⁴⁹³, AGS8-C2: A¹⁴⁹⁴– T¹⁵⁸⁵. (<i>lower panel</i>) GST-pulldown assay of AGS8 segments with Gβ₁γ₂. AGS8 domains synthesized as GST-fusion proteins (300 nM) were incubated with recombinant human Gβ₁γ₂ (30 nM) in a total volume of 300 µl at 4°C. Proteins were then adsorbed to a glutathione matrix and retained G-protein subunits identified by immunoblotting following gel electrophoresis. The representative of 4 independent experiments with similar results.</p

Release from complete replication blockage by a high concentration of aphidicolin or HU induces DSBs.

<p>(A) Cell-cycle analysis after treatment with 2 mM HU for 2 h. The BrdU-positive fraction was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g001" target="_blank">Figure 1</a>. (B) Frequency of chromosomal aberrations (CAs) for <i>wild-type</i> DT40 cells and <i>RAD54^−/−/KU70^−/−</i> cells. Cells were exposed to 2 mM HU for 2 h and then released in a drug-free medium for 3 or 6 h. (C) Cell-cycle analysis after treatment for 2 h with 0.5 µM aphidicolin (APH). The BrdU-positive fraction was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g001" target="_blank">Figure 1</a>. (D) Frequency of chromosomal aberrations (CAs) for <i>wild-type</i> DT40 cells and <i>RAD54^−/−/KU70^−/−</i> cells. Cells were exposed with 0.5 µM aphidicolin (APH) for 2 h and released in a drug-free medium for 3 or 6 h. More than 50 cells were analyzed in each case. Error bars show standard error for the number of CAs in 50 mitotic cells, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g002" target="_blank">Figure 2</a>. Asterisk and double asterisk: significant difference compared with <i>wild-type cells</i> (P<0.05).</p

Effect of deletion of 45 amino acids of fibronectin type 3 domain.

<p>(A) Schematic diagram of C-terminal of AGS8 (AGS8-C) and the deleted mutant of AGS8-C (delta AGS8-C) lacking the first 45 amino acids of fibronectin type 3 domain, that are A¹⁴⁹⁴ to R¹⁵³⁸ of rat AGS8 (ABB82299). (B) Bioactivity of AGS8C and delta AGS8C on G-protein activation. The yeast strain expressing human Gαs was transformed with AGS8-C and delta AGS8-C in the pYES2-containing GAL1 promoter. The yeast strain was modified to grow without histidine on activation of G-protein. Induction (+): induction of translation of AGS8 domains by galactose. The representative of 4 independent experiments with similar results.</p

Effect of AGS8-peptide on localization of connexin 43 (CX43) of cultured cardiomyocytes.

<p>(A) Cardiomyocytes were exposed 3 times to 30 min of hypoxia (1% oxygen) intermittent with 30 min of reoxygenation 4 h after (without or with) transfection of AGS8-peptide (1 µM) or FITC-conjugated AGS8-peptide (1 µM). (B) Localization of CX43 in the cardiomyocytes under normoxia and hypoxia. The figures demonstrated in the triple color of CX43 (<i>red</i>, <i>arrow</i>), nuclei (DAPI, <i>blue</i>) and FITC-conjugated AGS8-peptide (<i>green</i>). The representative of 5 independent experiments with similar results. (C) The number of cardiomyocytes expressing CX43 in the cell surface were counted. Please note that ∼ 90% cells were detectable at the point of fixation. Data are represent 170–260 cells from 5 of independent experiments. *, <i>p</i><0.05 vs normoxia group.</p