Additional file 5: Figure S2. of A radiosensitizing effect of RAD51 inhibition in glioblastoma stem-like cells

Representative comet images of A) GSC-11 (group 1) and B) GSC-14 (group 2) after RI-1 treatment and 16Gy IR (t = 180 min) Comet images were captured with the Axio Imager M2 fluorescent microscope (Carl Zeiss) at 20× (scale bar 100 μm). IR, Ionizing radiation (PPTX 280 kb

Clinopodium macranthum Hara

原著和名: ミヤマクルマバナ科名: シソ科 = Labiatae採集地: 長野県〜新潟県 雨飾山 (信濃〜越後 雨飾山)採集日: 1979/7/26採集者: 萩庭丈壽整理番号: JH018303国立科学博物館整理番号: TNS-VS-96830

Activities of proinflammatory cytokines on the differentiation of Reconstituted Human Epidermis.

<p>(A) RHE have been cultured for 10 days at the air-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM or TNFα alone or in combination during 72 h for immunohistological analysis. RHE were fixed, embedded in paraffin and 4 µm vertical sections were stained with Hematoxylin and Eosin (HE) or with anti-CK10, anti-LOR, anti-FLG, anti-IVL or anti-S100A7 mAbs. Results are from one experiment representative of two. (B) RHE have been cultured for 10 days at the air-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM and TNFα (3 ng/ml), with or without JAKs inhibitor (10 µM) during 72 h. RHE were fixed, embedded in paraffin and 4 µm vertical sections were stained with Hematoxylin and Eosin. Results are from one experiment representative of three.</p

Synergistic activity of proinflammatory cytokines on inhibition of KDM expression by NHEK.

<p>NHEK were cultured in the presence or absence of 10/ml of IL-1α, IL-17A, IL-22, OSM and TNFα alone or in combination for 24 h. Quantitative RT-PCR analysis was carried out on total RNA from 4 independent NHEK cultures. mRNA expression levels for cytokeratin 10 (CK10), cytokeratin 1 (CK1), desmoglein 1 (DSG1), desmocollin 1 (DSC1), fatty acid binding protein 5 (FABP5), calmodulin-like skin protein (CLSP), loricrin (LOR) and filaggrin (FLG) were normalized using GAPDH housekeeping gene and expressed as the fold decrease under unstimulated cultures. (A) Comparison of the activity of IL-1α, IL-17A, IL-22, OSM and TNFα alone or in combination (M5) on expression of keratinocyte differentiation markers. (B) Comparison of the activity of mix of 4 cytokines versus mix of 5 cytokines (M5) on expression of keratinocyte differentiation markers. All data are represented as mean and SEM of 4 independent experiments. One-way ANOVA with a Dunnett post-test were used for statistical evaluation and p values were as follows: *p<0.05, **p<0.01, ***p<0.001.</p

Synergistic activity of proinflammatory cytokines on inhibition of KDM expression by Reconstituted Human Epidermis.

<p>RHE have been cultured for 10-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM or TNFα alone or in combination during 24 h for mRNA quantification. Quantitative RT-PCR analysis was carried out and expression levels for KDM were normalized using GAPDH housekeeping gene and expressed as the fold to unstimulated control cultures. Data are mean and SEM of one experiment representative of two. One-way ANOVA with a Dunnett post-test were used for statistical evaluation and p values were as follows: *p<0.05, **p<0.01, ***p<0.001.</p

IL-22 activates the STAT3 signaling pathway in GBM cell lines.

<p>(A, B) The ability of IL-22 to activate signaling pathway in GBM cells was assessed using antibodies specific to STAT3 and Phospho-STAT3 (P-STAT3). U87MG (A) and U118MG (B) cells were stimulated with IL-22 and harvested at indicated times. Thirty mg of protein lysates was analyzed for P-STAT3 (Tyr705) and total STAT3 by western blot analysis. The density of each P-STAT3 band was corrected for variance in loading, using the density of the corresponding total STAT3. The expression level was evaluated as the ratio of phosphorylated STAT3 protein densities between control (0 min) and treated cells. Histograms are means ± SEM of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; when compared with control.</p

Exogenous IL-22 promotes cell survival of GBM cell lines.

<p>U87MG (A) and U118MG (B) cell lines were incubated for 24 to 48 h in basal culture medium (FCS 10%, +) and in serum-free medium (FCS 10%,-) in the presence of exogenous IL-22 (+) or without IL-22 (-). Cell proliferation was determined using BrdU cell proliferation assay. The data are represented as histograms of proliferating cells in relative units ± SEM of three independent experiments. *, a value of <i>p</i> < 0.05; when compared with respective control without exogenous IL-22. (C, D) Apoptotic ratios of soluble nucleosomes were detected by ELISA cell death for U87MG (C) and U118MG (D) cell lines induced after 48 h of incubation with Fas activation alone (Fas-L, +) or in combination with 20 ng/mL of recombinant IL-22 (IL-22, +). Histograms mean ratio of apoptotic cells ± SEM of three independent experiments. *, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; when compared with respective control. (E) The antiapoptotic factor Bcl-xL expression was assessed by western blotting (in reference to actin) in total cellular protein extracted from cells treated or not with Fas-L, with or without recombinant IL-22 for 48 h.</p

IL-22 induces STAT3 nuclear translocation and P-STAT3 nuclear accumulation in GBM cells.

<p>(A, B) Immunofluorescence analysis of STAT3 and P-STAT3 in U87MG (A) and U118MG (B) cells that were non-treated (NT) or treated with IL-22 for 30 minutes (IL-22). After the treatment, cells were fixed and stained with anti-STAT3 mouse mAb and anti-P-STAT3 rabbit mAb followed by Alexa fluor-conjugated fluorescent secondary antibody. Nuclei were counter stained with the blue-fluorescent DNA stain DAPI to point out nuclear localization of STAT3. Scale bars, 10μm.</p

IL-22 receptors are expressed in human GBM tumors.

<p>(A-C) Quantitative RT-PCR analysis of IL-22R (IL-22R1, IL-10R2) and IL-22 in total RNA extracted from 10 GBM-initiating cells established from GBM tumors (GL). Three independent NHEK cultures were used as a positive control for IL-22R1 (A) and IL-10R2 (B) mRNA expression and three human psoriatic skin biopsies (PSO) were used as a positive control for IL-22 (C). Target gene expression was normalized to the housekeeping GAPDH mRNA. (D) Detection of IL-22R1 and IL-10R2 proteins assessed by western blot analysis from two GBM-initiating cells. Positive control was HT29 cell line. Actin was used as a loading protein control.</p

IL-22 reduces ERK1/2 phosphorylation in GBM cell lines.

<p>(A, B) The expression of P-ERK1/2 and the total amount of ERK1/2 were analyzed by western blotting in total cellular protein extracted from U87MG (A) and U118MG (B) cells treated with IL-22 for the indicated times. Thirty mg of protein lysates was analyzed for P-ERK1/2 and total ERK1/2. The density of each P-ERK1/2 band was corrected for variance in loading, using the density of the corresponding total ERK1/2. The expression level was evaluated as the ratio of phosphorylated ERK1/2 protein densities between control (0 min) and treated cells. A representative results of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; when compared with control. (C, D) Effect of U0126 on proliferation of GBM cells. BrdU cell proliferation assays of U87MG (C) and U118MG (D) cells treated for 24 h in serum-free medium with vehicle (non-treated; NT) or with 0.5 and 5 μM of U0126. The data are represented as histograms of proliferating cells in relative units. Error bars indicate ± SEM. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01.</p