Boletín de Segovia: Número 1 - 1837 enero 3

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 2: Figure S1. of Extracellular nucleotides as novel, underappreciated pro-metastatic factors that stimulate purinergic signaling in human lung cancer cells

Evaluation of the number of BM cells and the level of EXN in plasma after irradiation or chemotheraphy. Panel A Total number of cells isolated from tibia and femurs of animals 24 h after irradiation (0–1500 cGy) or vincristine administration (0.5–2 mg/kg). Combine results from three independent isolations. Panel B The level of ATP, UTP and adenosine in murine plasma isolated from animals 24 h after irradiation (0–1500 cGy) or vincristine administration (0.5–2 mg/kg). (PDF 229 kb

Immunofluorescence studies of P2X7 receptor and differentiation-stage specific proteins in undifferentiated and differentiated ESC.

<p>P2X7R expression and co-localization with differentiation-stage specific proteins was determined by confocal microscopy and immunofluorescence assays as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096281#s2" target="_blank">Methods</a>. (<b>A</b>). Left panel: Co-localization between SSEA-1 (stage-specific embryonic antigen-1) and P2X7R immunofluorescence in undifferentiated ES cells. Co-localization of the protein is shown by Z-stack analysis. Right panel: Double-immunostaining for nestin (neural stem and precursor marker) and P2X7R expression in cells induced to differentiation for 8 days. (<b>B</b>). Left panel: Immunostaining for Oct-4 (pluripotency marker) in undifferentiated cells. Middle panel: Immunostaining for Oct-4 and SSEA-1 in undifferentiated cells. Right panel: Staining pattern for the neuron-specific marker β-tubulin in neural-differentiated cells. Cell nuclei were visualized by DAPI staining. Scale bar, 50 µm.</p

Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

<div><p>Background</p><p>Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.</p><p>Principal Findings</p><p>P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.</p><p>Conclusions</p><p>In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.</p></div

Expression of the proliferation antigen Ki-67 in SSEA-1 positive cells differentiated in the absence or presence of P2X7R agonists and antagonists.

<p>(<b>A</b>) Flow cytometry determination of Ki67⁺ cells following neural differentiation for 8 days in the absence or presence of the agonist Bz-ATP (10 µM), the antagonists KN-62 (10 µM) or A438079 (1 µM), or Bz-ATP and A438079. (<b>B</b>) Determination of percentages of Ki67⁻/SSEA-1⁺ and Ki67⁺/SSEA-1⁺ cells for experimental conditions explained in A. (<b>C</b>) Representative dot-plot images for Ki67/SSEA-1 double staining for conditions described in B. Statistical relevance was analyzed by the One-Way ANOVA test followed by the Bonferroni post hoc test. Bars represent mean ± SE of 5 independent experiments (*p<0,05, ***p<0.001 compared to control data).</p

Inhibition of embryonic stem cell growth in the presence of P2X7R inhibitors.

<p>Cells were plated at 3X10⁵ cell/ml density and P2X7R was inhibited daily with 10 µM KN-62 or 1 µM A438079 and then the number of cells were counted. Data represent mean values ±S.E. of three independent experiments performed in triplicate (*p<0,05 compared to control).</p

Proliferation of ESC in conditions of P2X7R modulation.

<p>Cell cycle analysis based on flow cytometric analysis of BrdU incorporation and propidium iodide DNA-staining were performed as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096281#s2" target="_blank">Methods</a>. (<b>A</b>) Cell distributions at different S-phases of ESC treated with P2X7R agonist or inhibitor, 0.1 µM and 1 µM Bz-ATP and 10 µM KN-62 for 96 h, respectively. Shown data are representative for mean values ±S.E. of five independent experiments. Data were statistical analyzed by the One-Way ANOVA test followed by the Bonferroni post hoc test with (*p<0,05 compared to control data). (<b>B</b>) Representative BrdU/PI cell cycle analysis of ESC treated with Bz-ATP or KN-62 compared to untreated control cultures. (<b>C</b>). Cell cycle distributions of ESC treated for 96 h with the P2X7R agonist (0.1 µM or 1 µM Bz-ATP) or the P2X7R inhibitor KN-62 (10 µM), respectively. Shown data are representative for mean values ±S.E. of five independent experiments.</p

P2X7 receptor and Oct-4 expression in undifferentiated mouse ESC and cells induced to neural differentiation.

<p>P2X7 receptor and Oct-4 expression levels in undifferentiated (und) and cells induced to differentiation (days 0–8) were determined by (<b>A</b>) real-time PCR and (<b>B,C</b>) Western blotting assays as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096281#s2" target="_blank">Methods</a>. For real-time PCR, quantitative analysis of the relative expression of P2X7R and Oct-4 in E14Tg2A cell line was performed using GAPDH mRNA transcription rates as endogenous control for normalization of expression levels. For Western blotting, P2X7R and Oct-4 expression levels were obtained and analyzed by densimetric analysis of protein bands and were compared to β-actin expression levels. The P2X7R was identified by two antibodies, which recognize the extracellular or C-terminus domain. Bars represent mean ± standard errors (S.E.) of three independent experiments performed in triplicate. Data were analyzed for statistical relevance with the One-Way ANOVA test followed by the Bonferroni post hoc test (*p<0,05,**p<0,01, ***p<0.001 compared to control data).</p

Modulation of neuroectodermal differentiation of mouse ESC by P2X7R activity.

<p>(<b>A</b>) Flow cytometry analysis of SSEA-1 and β3-tubulin protein expression in neural-differentiated ES cells. Cells were cultured and induced to differentiation as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096281#s2" target="_blank">Methods</a>. (<b>B</b>) Immunofluorescence assay of SSEA-1 and β3-tubulin expression of cells following 8 days of differentiation. Circled areas: <b>1</b>. cells expressing only SSEA-1, <b>2</b>. cells co-expressing SSEA-1 and β3-tubulin. Scale bar, 50 µm. (<b>C</b>) Flow cytometry analysis of SSEA-1-positive cells co-expressing or not β3-tubulin on day 8 of differentiation cultured in the presence of 10 µM Bz-ATP or 10 µM KN-62, respectively. (<b>D</b>) Percentage of β3-tubulin-positive cells differentiated in the absence or presence of 1 µM A438079 or 10 µM KN-62, as determined by flow cytometry. Statistical relevance was analyzed by the One-Way ANOVA test followed by the Bonferroni post-hoc test. Six independent experiments were performed (* p<0.05 compared to control data).</p

Differential expression of P2X7R alternative splicing isoforms in undifferentiated and neural- differentiated ESC.

<p>(<b>A</b>) P2X7 receptor isoform A, B, C and k expression in undifferentiated (UND) and cells following 8 days of neural differentiation (DIFF) was determined by RT-PCR. (<b>B</b>) P2X7R isoforms expression levels were obtained and analyzed by densimetric analysis of DNA bands and were compared to GAPDH expression levels. Data were analyzed for statistical relevance with the One-Way ANOVA test followed by the Bonferroni post hoc test (*p<0,05,**p<0,01, ***p<0.001 compared to control data).</p