Effect of hypoxia on the activation kinetics and voltage dependence of activation of outward currents.

<p>(A) The mean normalized conductance data are presented for normoxic (n = 53) and hypoxic cultures (n = 47). To obtain the steady-state activation curves, the data were fitted using the Boltzmann sigmoid equation. Hypoxic culture shifted the steady-state activation to the left, indicating that lower voltages were required to open the channels, but the slope factor <i>k</i> was not changed. The potential where activation reached 50% of its maximum (<i>V_0.5</i>) is indicated. (B) The graph shows the average ratio between currents at 20 mV and 60 mV for normoxic and hypoxic cultures. The asterisk (*) denotes a statistically significant difference (p<0.05). (C) Frequency distribution histograms for the 20 mV/60 mV current ratios. Data were fitted using Gaussian distribution functions.</p

Ion channel gene expression in response to hypoxia.

<p>(A) Transcriptional expression of selected outward and inward ion channel genes (Kv1.1, Kv2.1, Kv3.4, Kv4.3, Kv7.3, MaxiK, HCN2, Kir2.1, and α1C) was assessed after 4 days of culture in standard normoxic conditions and in hypoxia. (B) Expression of MaxiK, HCN2 and α1C, genes during 13 days of culture in 20% and 5% oxygen concentration. The data were obtained from repeated experiments using all three cell cultures (n = 6). The asterisks (*) denote a statistically significant difference (p<0.05) with respect to D0.</p

Inhibition of outward currents by TEA in ASCs cultured in normoxia and hypoxia.

<p>(A) Reduction in the steady-state current values as a result of 10 min perfusion with 20 mM TEA. Asterisks (*) denote a statistically significant difference (p<0.05) between the control and TEA-treated cells (n = 6). (B) TEA-mediated shift of the steady-state activation curves. The mean normalized conductance data was fitted using the Boltzmann sigmoid equation. (C) Boxplot (middle line: median; box: upper and lower quartile; bars: minimum and maximum values) of the change in <i>V_0.5</i> after addition of TEA. No statistical significant differences were found (Mann-Whitney U test).</p

Growth and dose-dependent growth inhibition of ASCs in normoxia and hypoxia.

<p>(A) Cell numbers were greatly increased under hypoxic conditions after 4 days in culture for all three cell cultures (n = 6). Asterisk denotes statistically significant difference (p<0.01). (B) Dose-dependent growth inhibition of ASCs by TEA. The data were obtained from repeated experiments using all three cell cultures (n = 6) and was normalized to the number of cells in normoxic cultures without TEA. The half maximal inhibitory concentration (IC₅₀) is indicated.</p

Additional file 4: Table S3. of Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix

Proteins in the peptidome fraction regulated by hypoxia. (DOCX 16 kb

Additional file 1: of Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS)

List of antibodies. (DOCX 12 kb

Stabilisation of HIF-1 in hASCs after trypsin and 1% oxygen exposure alone or in combination.

<p>(<b>A</b>) HIF-1 activation/stabilization during 6 hours culture after trypsin exposure in combination with 24 hours in hypoxic/normoxic conditions was analysed by ELISA. All cells were harvested <i>in situ</i>. Values are represented as the mean and SEM (n = 12). Asterisks denote statistical difference between this and all other groups (p<0.05). (<b>B</b>) Analysis of HIF-1α induction at 4 and 12 hours following 5 min trypsin exposure was done by immunoblotting. All cells were harvested <i>in situ</i>. HIF-1α positive controls are ASCs subjected to 48 hours of 1% oxygen.</p

Trypsin-activated PAR2 intracellular signaling in hASCs.

<p>(<b>A</b>) Schematic rendition of signal-transduction pathways linking PAR2 and <i>VEGF</i>. (<b>B</b>) The effect of specific kinase inhibitors on suppressing trypsin-induced <i>VEGF</i> activation after 5 min trypsin exposure was assessed by real-time RT-PCR (n = 6). Expression levels were normalized to the levels induced by trypsin (Ctrl). (<b>C</b>) The effect of PI3K and Mek inhibitors on phosphorylation of Akt and Erk1/2, respectively, as a result of 5 min trypsin exposure was determined by immunoblotting. PI3K and Mek inhibitors were added 2 hours prior to trypsin exposure. Cells after a 4-day culture at 20% oxygen were used as controls (Ctrl). Representative data obtained from ASC12 cells are presented. Values are represented as the mean and SEM. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control.</p

Expression of PAR2 in hASCs and its association with transcriptional activation of <i>VEGF</i>.

<p>(<b>A</b>) Detection of PAR2 in hASC lines by immunoblotting. (<b>B</b>) Expression of PAR2 by indirect immunofluorescence using SAM11 antibody. Representative pattern as detected on the surface of ASC12 cells is presented. (<b>C</b>) SAM11 antibody blocked trypsin-induced PAR2 activation, measured using real-time RT-PCR to determine <i>VEGF</i> expression levels 12 hours after trypsin exposure (n = 15). Expression levels were corrected for basal <i>VEGF</i> activity in hASCs cultured at 20% oxygen and normalised to the levels induced by trypsin. Values are represented as the mean and SEM. Scale bar indicates 200 µm. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control (NIH 3T3 cells).</p

Immunophenotypical analysis of hASC lines at passage 2.

<p>(<b>A</b>) Representative distributions of positive markers expressed on the ASC12 cells are presented. (<b>B</b>) Surface markers profile was obtained as an average from ASC12, 21, and 23 lines.</p