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Additional Supporting Information may be found in the online version of this article.

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sc-12-0820_sm_SupplFigure1.tif2814KSupplementary Figure S1. (A): Immunofluorescence staining (red) of TS and TS-RR for glial fibrillary acid protein (GFAP, astrocytic marker), Tuj1 (neuronal marker), and NG2 (oligodendrocytic marker) after culture of the cells over 2 weeks in DMEM-F12 supplemented with 10% fetal calf serum (FCS). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. (B): Doubling time of TS and TS-RR. Data are means ± SD from three independent experiments. *P < 0.05. (C): Flow cytometric analysis of cell cycle for TS and TS-RR cells. (D): Flow cytometric analysis of G0 phase cells for TS and TS-RR cells detected by pyronin Y/Hoechst staining. Percentages of G0 phase cells are indicated in red.
sc-12-0820_sm_SupplFigure2.tif613KSupplementary Figure S2. (A): Flow cytometric analysis of cell cycle status for TS and TS-RR cells before (control), as well as 24 or 48 h after exposure to 5 Gy of ionizing radiation. (B): Quantification of the sub-G1 (apoptotic) cell population for the experiment in (A).
sc-12-0820_sm_SupplFigure3.pdf485KSupplementary Figure S3. Clonogenic survival assay for TS and TS-RR clones subjected (or not, control) to ionizing radiation with a single dose of 5 Gy as in Figure 3. Representative images of colonies formed 13 days after irradiation are shown. Scale bars, 10 mm.
sc-12-0820_sm_SupplFigure4.tif882KSupplementary Figure S4. (A): Quantification of relative expression levels of ICAM-1, osteopontin, IGFBP2, bFGF and lungkine by use of the cytokine array in Figure 4C. (B): Immunoblot analysis of the insulin receptor, FoxO1, and FoxO4 in TS exposed to ionizing radiation (5 Gy) for the indicated number of times.
sc-12-0820_sm_SupplFigure5.tif879KSupplementary Figure S5. (A): Quantitative analysis of nucleus-cytoplasm ratio (N/C) of FoxO3a in experiments similar to that in (Fig. 5A). Data are means ± SD from three independent experiments. *P < 0.05. (B): Quantitative analysis of PKH26 positive cells 4 days after staining. Data are means ± SD from three independent experiments. *P < 0.05. (C): Flow cytometric analysis of cell cycle status for TS and TS-RR cells treated with control or FoxO3a shRNAs. (D): Quantification of each cell population for the experiment in (C).
sc-12-0820_sm_SupplFigure6.pdf423KSupplementary Figure S6. (A): The phosphorylation patterns of IGF1R analyzed by phos-tag immunoblotting in TS with or without irradiation and neutralizing antibody for IGF1. (B): The phosphorylation patterns of IGF1R analyzed by phos-tag immunoblotting in TS-RR with or without irradiation and neutralizing antibody for IGF1. (C): Colony formation for TS and TS-RR after exposure to a single dose (4 Gy) of ionizing radiation and addition of IGF1 to growth factor–depleted medium. Scale bar, 10 mm. (D): Self-renewal ability of TS-RR and TS after long-term treatment with IGF1 (50ng/ml), as evaluated with sphere formation assays. Scale bar, 10 mm. (E): Clonogenic survival assay for IGF1-treated TS and TS-RR after exposure to 5 Gy of ionizing radiation. Scale bar, 10 mm.
sc-12-0820_sm_SupplFigure7.pdf546KSupplementary Figure S7. (A): Cell survival analysis for TS and TS-RR 48 hours after addition of PPP as evaluated with WST-8 assays. Data represent cell survival relative to untreated controls for each cell type and are means ± SD from three independent experiments. *P < 0.05 (treated vs. control). (B): The phosphorylation patterns of IGF1R analyzed by phos-tag immunoblotting in TS 30 minutes after addition of 50ng/ml of IGF1 or 1 hour after addition of PPP (0.75μM). (C): Immunoblot analysis of IGF1 downstream factors in TS 30 minutes after addition of 50ng/ml of IGF1 or 1 hour after addition of PPP (0.75μM). (D): Immunoblot analysis of cytoplasmic and nuclear fractions of TS and TS-RR with or without irradiation and addition of PPP (0.6μM) at the time of 6 hours after irradiation. Internal controls: MEK1/2 - cytoplasmic fraction, lamin c - nuclear fraction. (E): Colony formation for TS and TS-RR after exposure to a single dose (4 Gy) of ionizing radiation together with either PPP (0.4 μM) or DMSO (control). Scale bar, 10 mm. (F): Colony formation for TS and TS-RR after exposure to a single dose (4 Gy) of ionizing radiation together with either AEW541 (2.0 μM) or DMSO (control). Scale bar, 10 mm. (G): Quantification of colony number in experiments similar to that in (F). Data are expressed as the fraction of surviving cells for TS or TS-RR relative to the corresponding control value and are means ± SD from three independent experiments. *P < 0.05. (H): Immunofluorescence staining (red) of phospho-IGF1R (Tyr1161) in tumors formed by TS-RR (day14), with and without PPP treatment. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm.
sc-12-0820_sm_SupplInfor.pdf98KSupplementary Data

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