Study schematic and immuno-phenotype of ASCs.

<p>Panel (a) Schematic of the <i>in vivo</i> and <i>in vitro</i> experiment. Panel (b) CD29, CD31, CD44 and CD45 were detected by flow cytometry. Results showed that the fourth passage ASCs were largely positive for CD29 (99.80±0.10%) and CD44 (99.60±0.20%), and only minority of ASCs were positive for CD31 (0.30±0.10%) and CD45 (0.45±0.10%). The expression of CXCR4 on ASCs was 9.96±0.07%.</p

Comparisons of eNOS, p-eNOS, NO and SDF-1α levels in each group.

<p>Panel (a) Western-blot analysis showed that a significant increment of eNOS expression in group Ator, A+L-NAME and A+AMD3100 for <i>in vivo</i> study when compared with blank control. (sham operated: 0.28±0.07, blank control: 0.41±0.10*, Ator: 0.88±0.16*#, A+L-NAME: 0.93±0.15*#, A+AMD3100: 0.92±0.13*#). Similar changes were found in p-eNOS expression among each group (sham operated: 0.33±0.06, blank control: 0.46±0.11*, Ator: 0.93±0.14*#, A+L-NAME: 1.01±0.18*#, A+AMD3100: 0.95±0.16*#). Panel (b) Analyses of eNOS expression by Western-blot for <i>in vitro</i> study showed that as compared with group blank control, eNOS was significantly increased in group Ator, A+L-NAME and A+AMD3100. (sham operated: 0.42±0.08, blank control: 0.61±0.10*, Ator: 0.86±0.13*#, A+L-NAME: 0.89±0.11*#, A+AMD3100: 0.92±0.14*#). Similar changes were found in p-eNOS expression among each group (sham operated: 0.33±0.06, blank control: 0.50±0.09*, Ator: 0.76±0.11*#, A+L-NAME: 0.81±0.13*#, A+AMD3100: 0.78±0.10*#). Panel(c) When compared with group blank control, the NO productions <i>in vitro</i> and <i>in vivo</i> were identically and significantly increased in group Ator and A+AMD3100, whereas was abolished in group A+L-NAME. (<i>In vivo</i> study, sham operated: 6.03±1.10, blank control: 6.22±0.98, Ator: 10.31±0.86*#, A+L-NAME: 8.86±1.03*#, A+AMD3100: 11.10±0.92*#) and (in vitro study, sham operated: 3.62±0.42, blank control: 4.11±0.66, Ator: 7.03±0.71*#, A+L-NAME: 5.02±0.69*#, A+AMD3100: 7.11±0.70*#). Panel (d) As compared with group blank control, the SDF-1α expressions <i>in vitro</i> and <i>in vivo</i> was profoundly increased with atorvastatin therapy; however, the effects were reduced in group A+L-NAME. (<i>In vivo</i> study, sham operated: 5.53±0.73, blank control: 6.05±0.95, Ator: 10.88±1.33*#, A+L-NAME: 7.92±1.09*#, A+AMD3100: 9.86±1.21*#) and (in vitro study, sham operated: 3.84±0.47, blank control: 4.21±0.65, Ator: 8.12±0.77*#, A+L-NAME: 5.88±0.70*#, A+AMD3100: 7.76±0.73*#).</p

Comparison of ASCs apoptotic index <i>in vivo.</i>

<p><i> In vivo</i> apoptotic index was detected by TUNEL, and the results showed that compared with group blank control, atorvastatin therapy significantly reduced apoptotic index of ASCs, nevertheless, the efficacy was offset when either L-NAME or AMD3100 was added. (sham operated: 22.6±5.5, blank control: 52.1±9.4*, Ator: 31.3±6.7*#, A+L-NAME: 46.0±8.3*$, A+AMD3100: 49.7±7.8*$, per high power field). (Scale Bar: 50 µm).</p

Evaluation of ASCs migration and differentiation <i>in vivo</i>.

<p>Seven days after ASCs transplantation, immuno-staining analysis showed that in comparison of group blank control, the number of ASCs migrated in peri-infarcted areas was significantly increased in group Ator, however, the efficacy with atorvastatin therapy was abolished when SDF-1α up-regulation was diminished or SDF-1α/CXCR-4 coupling was blockage as shown in group A+L-NAME and A+AMD3100. (sham operated: 4.4±2.3, blank control: 13.3±2.2*, Ator: 22.6±2.9*#, A+L-NAME: 15.0±2.3*$, A+AMD3100: 14.5±2.5*$, per high power field). No significant difference was found in ASCs differentiation among each group (sham operated: 4.6±0.2%, blank control: 4.7±0.2%, Ator: 4.5±0.2%, A+L-NAME: 4.7±0.2%, A+AMD3100: 4.6±0.2%). (Scale Bar: 50 µm).</p

CoCl₂-induced expression of HIF-1α in PC12 cells.

<p>(A) time course for the effects of CoCl₂ on expression of HIF-1α detected by western blot analysis; (B) Denstiometric analysis for the results in (A) with the Image J 1.41o software. Data were shown as the mean ± SE (n = 3). ^##<i>P</i><0.01 compared with control group (0h).</p

Effects of NaHS and NAC on CoCl₂-induced overproduction of reactive oxygen species (ROS) in PC12 cells.

<p>(A) Random micrographs of dichlorofluorescein (DCF)-derived fluorescence in PC12 cells. A-a: Control, untreated cells; A-b: NaHS group, cells were treated with 400 µmol/L NaHS for 30 min alone; A-c: NAC group, cells were treated with 500 µmol/L NAC for 60 min alone; A-d: CoCl₂-treated group, CoCl₂ cells treated with 600 µmol/L CoCl₂ for 6 h; A-e: NaHS+CoCl₂ group, cells were preconditioned with 400 µmol/L NaHS for 30min prior to treatment with 600 µmol/L CoCl₂ for 6 h; A-f: NAC+CoCl₂ group, cells were preconditioned with 500 µmol/L NAC for 60 min prior to treatment with 600 µmol/L CoCl₂ for 6 h; (B) Quantitative analysis of the mean fluorescence intensity in the indicated groups. Data are the mean ± SE (n = 3).^##<i>P</i><0.01 compared with control, **<i>P</i><0.01 compared with CoCl₂-treated group.</p

Kinase inhibitors suppressed CoCl₂-induced expression of cleaved-caspase-3.

<p>(A) time course for the effects of CoCl₂ on expression of cleaved-casepase-3. (A-b) Denstiometric analysis for the results in (A-a). Data are presented as the mean ± SD from independent experiments performed in triplicate. ^#<i>P</i><0.05,^##<i>P</i><0.01 compared to the control group. (B) and (C) PC12 cells were preconditioned with 10 µmol/L MEK1/2 (upstream of ERK1/2) inhibitor U0126 for 120 min (B) and 20 µmol/L p38MAPK inhibitor SB203580 for 60 min (C) before exposure of cells to 600 µmol/L CoCl₂ for 9 h. Panels B-b and C-b show denstiometric analysis for the data from B-a or C-a, respectively. Data are presented as mean ± SE from independent experiments preformed in triplicate. ^##<i>P</i><0.01 compared to the control group, **<i>P</i><0.01 compared to the CoCl₂ group.</p

NaHS attenuated CoCl₂-induced ERK1/2 and p38 MAPK phosphorylation in PC12 cells.

<p>PC12 cells were preconditioned with 400 µmol/L NaHS for 30min before exposure of cells to 600 µmol/L CoCl₂ for 30 min (A) and (B) or for 120min (C) and (D). Cell lysates were subjected to western blot analysis using anti-p-ERK1/2 and anti-ERK antibody (A and B) or anti-p-p38 and anti-p38 antibody (C) and (D). Panels (B) and (D) show denstiometric analysis for the data from (A) or (C), respectively. Data are presented as mean ± SE from independent experiments preformed in triplicate. ^##<i>P</i><0.01 compared to the control group, **<i>P</i><0.01 compared to the CoCl₂ group.</p

NAC attenuated CoCl₂-induced ERK1/2 and p38MAPK phosphorylation in PC12 cells.

<p>PC12 cells were pretreated with 500 µmol/L NAC for 60 min prior to exposure of cells to 600 µmol/L CoCl₂ for 30 min (A) and (B) or 120 min (C) and (D). Cell lysates were subjected to western blot analysis using anti-p-ERK1/2 and anti-ERK1/2 antibody (A) and (B) or anti-p-p38 and anti-p38 antibody (C) and (D). (B) and (D) show denstiometric analysis for the data from (A) or (C), respectively. Data are presented as mean ± SE from independent experiments preformed in triplicate. ^##<i>P</i><0.01 compared to the control group, **<i>P</i><0.01 compared to the CoCl₂ group.</p

ERK1/2, p38MAPK and JNK pathways mediated CoCl₂-induced injuries in PC12 cells.

<p>(A) and (B) The cell viability was assessed by the CCK-8 reduction method described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025921#s2" target="_blank">‘Materials and methods’</a>. The cells were treated with the indicated treatments. (C) Morphological changes in apoptotic cells assessed by Hoechst 33258 staining. Arrow indicates cells with apoptotic nuclear condensation and fragmentation. Control: untreated cells; SB203580: cells were treated with 20 µmol/L SB203580 for 60 min alone; UO126: cells were treated with 10 µmol/L UO126 for 120min alone; CoCl₂: cells were treated with 600 µmol/L CoCl₂ for 48 h; SB203580+ CoCl₂: cells were pretreated with p38 inhibitor SB203580 (20 µmol/L) for 60 min followed by exposure of cells to 600 µmol/L CoCl₂ for 48 h; UO126+ CoCl₂: cells were pretreated with ERK1/2 inhibitor UO126 (10 µmol/L) for 120 min followed by exposure of cells to 600 µmol/L CoCl₂ for 48 h. (D) Results from FCM analysis. Data are the mean ± SE (n = 3).^##<i>P</i><0.01 compared with control; **<i>P</i><0.01 compared with CoCl₂-treated group.</p