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

Long-term exposure to nucleoside analogues increased mtDNA loss of mouse cerebral cortical neurons.

<p>A. Typical images of Laser Capture Microdissection before and after microdissection (×200 to ×400 magnification). B. Compared with control, mtDNA significantly decreased in captured neurons from brain tisssues of the mice treated with D4T (50 mg/kg), AZT (100 mg/kg), 3TC (50 mg/kg) and DDI (50 mg/kg) for four months as measured by COXII specific primers and probes (“*” denoted <i>p</i><0.05, “**” denoted <i>p</i><0.01).</p

Long-term nucleoside analogue exposures mainly induced mouse neuronal mtDNA major arc deletion.

<p>A. The number of captured neuron ND4 (mtDNA major arc) copies significantly decreased in the mice treated with D4T (50 mg/kg), AZT (100 mg/kg), 3TC (50 mg/kg) or DDI (50 mg/kg) for four months. But only AZT group had a decline in ND1 (mtDNA minor arc) copies. B. The numbers of ND4 and ND1 copies from captured glial cells had no significant changes in all four–month-NRTI-treatment groups except for D4T group in which the number of ND4 copies increased instead of reduction (“*” denoted <i>p</i><0.05, “**” denoted <i>p</i><0.01).</p

Long-term exposure to nucleoside analogues did not affect total mtDNA levels of mouse cerebral cortex as measured by COXII specific primers and probes.

<p>Compared with control, total mtDNA copies significantly decreased in livers of the mice treated with D4T (50 mg/kg), AZT (100 mg/kg) and DDI (50 mg/kg) for four months, but total mtDNA did not change in livers of the mice treated with 3TC (50 mg/kg) for four months. Total mtDNA significantly decreased in muscles of the mice treated with D4T (50 mg/kg), AZT (100 mg/kg), 3TC (50 mg/kg) and DDI (50 mg/kg) for four months. But total mtDNA copies did not lose in brain tisssues of the mice treated with DDI, D4T, AZT and 3TC for four months. Further, mtDNA had even compensatory increased in D4T group and 3TC group (“*” denoted <i>p</i><0.05, “**” denoted <i>p</i><0.01).</p