T treatment reduced the aggregates or diffusion of cathepsin B at six h (Figure 4) or cathepsin L at 3 h (Figure five) post-OGD. We additional tested the effects of 3-MA on OGD-induced activation of caspase-3 in astrocytes with immunostaining. The results showed that significantly less active caspase-3 immunoreactivity was observed in non-OGD astrocytes (Supplementary Figure S5). In astrocytes treated with OGD, the active caspase-3-positive astrocytes Liquiritin MedChemExpress elevated with time and peaked at 12 h immediately after OGD (Supplementary Figure S5). In contrast, 3-MA reduced active caspase-3-positive astrocytes at 12 h just after OGD (Figure 6). In addition, we confirmed the role of caspase-3, z-VAD-fmk (nonspecific caspase inhibitor) and Q-DEVD-OPh (a distinct inhibitor of caspase-3) both decreased the protein levels of caspase-3 (Supplementary Figures S6a and c, b and d), suggesting that caspase-3 is activated in our OGD model system. To additional confirm the function of caspase-3, the LDH leakage was measured. Each z-VAD-fmk and Q-DEVD-OPh at 25 and 50 M markedly decreased the leakage of LDH in astrocytes 12 h post-OGD (Supplementary Figures S6e andg, f and h), indicating that inhibition of caspases or caspase-3 features a protective effects on ischemic astrocytes. These information additional recommend that the protective effects of autophagy inhibition on ischemic astrocytes are potentially mediated by inhibiting the activation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21338096 of caspase-3. Inhibition of autophagy decreases OGD-induced LMP in astrocytes. Excessive autophagy induces LMP35,36 and it is actually feasible that LMP mediates cathepsin B and L cytosolic translocation. Hence, we evaluated LMP formation by Acridine Orange (AO) and Lyso-Tracker Red staining assays. Ordinarily, AO, a metachromatic fluorophore cloistering inside on the lysosome, exhibits a higher level of red fluorescence and a low amount of green fluorescence. When lysosomes are disrupted, AO relocates for the cytosol from the lysosomes and manifests a lowered red fluorescence and an enhanced green fluorescence.36 As shown in Figures 7a and c, OGD induced a reduction in red fluorescence in astrocytes. In contrast, treatment with 3-MA or Wort markedly inhibited OGD-induced reduction in red granular fluorescence of AO staining. Lyso-Tracker Red uptake photos in astrocytesFigure 6 The therapy of 3-MA inhibits OGD-induced activation of caspase-3 in astrocytes. (a) Astrocytes had been treated with 3-MA (1 mM) and underwent OGD therapy for 12 h, after which the double immunofluorescence staining of caspase-3 (green) and GFAP (red) in astrocytes was performed by corresponding antibodies. DAPI (blue) was used to stain nuclei. Images had been captured by the confocal microscopy. Magnified photos (M) were cropped sections from the merge pictures (white borders). Magnification 200. (b) Quantification of active capase-3-positive cells as a percentage of total GFAP-positive cells. Signifies S.D., n = three. Po0.01 versus non-OGD group; Po0.01 versus OGD groupCell Death and DiseaseAutophagy inhibition blocks cathepsins release X-Y Zhou et alFigure 7 Inhibition of autophagy decreases LMP in OGD-treated astrocytes with AO-uptake and Lyso-Tracker Red uptake approaches. (a and b) Representative photomicrographs of AO staining (a) or Lyso-Tracker Red staining (b). Cells have been treated with OGD for 6 h, and then incubated with AO (five gml) for 15 min or Lyso-Tracker Red (75 nM) for 60 min. 3-MA (1 mM) or Wort (100 nM) was added in cells 30 min or two h ahead of OGD, respectively. The photos had been captured by a confocal microscope. Magnified.