Representative immunofluorescence of retinal sections showing the expression pattern of GLAST following ischemia.

<p>Glast immunoreactivity in the ischemic retina after 150 min of reperfusion (ISCH/REP) shows no evident changes in the expression and distribution of the glial transporter compared to the contralateral non-ischemic retina (CTL). (GCL) ganglion cell layer (IPL), inner plexiform layer, (INL) inner nuclear layer, (OPL) outer plexiform layer, (ONL) outer nuclear layer.</p

Confocal microscopy of DAPI-stained SH-SY5Y cells treated for up to 1 h with BEO.

<p>SH-SY5Y cells were exposed to the indicated concentration of BEO (0.005–0.02%) for 30 or 60 minutes. Apoptotic nuclear morphological changes, such as chromatin condensation and marginalization (arrows), are evident in cells exposed to BEO 0.02%, whereas no nuclear alterations are present in cells exposed to lower concentrations (0.005–0.01%). Images are representative of three independent experiments.</p

Native and bi-dimensional western blotting analysis of GLT-1.

<p>(A) GLT-1 immunoreactivity is increased under native conditions in the ischemic retina (I/R) after 150 min of reperfusion when compared to the non-ischemic contralateral eye (C). Histogram shows the results of the densitometric analysis of autoradiographic bands normalized to the value of loading control. Each value is the mean ± S.E.M. of three experiments. (B) A representative Native/SDS-PAGE image showing the appearance in the second dimension (SDS-PAGE) of a 38 kDa band up-regulated in the retina subjected to 50 min of ischemia and 150 min reperfusion (ISCH/REP) as compared to the contralateral non-ischemic retina (CTL).</p

Modulation of [³H]-D-Aspartate release and uptake in synaptosomes from ischemic retina.

<p>(A) <i>Ischemia reduced [³H]-D-Asp uptake in highly pure nerve ending.</i> [³H]-D-Asp uptake into synaptosomes was measured in animals subjected to retinal ischemia for 50 min in the right eye and reperfused for 150 min (dashed line). The contralateral eye (solid line) was used as control. Data are expressed as mean ± S.E.M. (n = 3 or 4 animals per group). (B) <i>[³H]-D-Asp release in retinal synaptosomes.</i> Animals were subjected to retinal ischemia for 50 min in the right eye and reperfusion was allowed for 150 min. Release was measured in the ischemic (white histograms), contralateral (grey) and naïve retinas (black). All results are expressed as percentage of the corresponding KCl-evoked release (15 mM). Omission of Ca²⁺ produces a reduction of [³H]-D-Asp release of about 40% in all groups, while DL-TBOA reduces by about 60% the release in naïve and contralateral samples, but not in the ischemic retina. *p<0.05 vs control.</p

Effects of retinal ischemia/reperfusion on the expression and distribution of glutamate transporter GLT-1.

<p>GLT-1 immunoreactivity increases in the retina of rats subjected to 50 min of ischemia followed by 150 min of reperfusion (ISCH/REP) as compared to the control, non-ischemic retina (CTL). An increased number of GLT-1 positive cell bodies is evident in the ONL and INL of retinas subjected to ischemia (ISCH/REP). (GCL) ganglion cell layer (IPL), inner plexiform layer, (INL) inner nuclear layer, (OPL) outer plexiform layer, (ONL) outer nuclear layer.</p

BEO activates autophagy in SH-SY5Y cells.

<p>(<b>A</b>) Concentration-dependent induction of autophagy by BEO. Representative immunoblot showing LC3I to LC3II conversion and reduced p62 levels following exposure of SH-SY5Y to increasing concentrations of BEO. Cells were incubated with medium containing either vehicle (ethanol, 0.0045–0.027%) or BEO (0.005–0.03%) for 1 h. GAPDH was used as loading control. LC3II/LC3I optical density (OD) ratio for the reported blot is shown. Histogram shows the results of densitometric analysis of p62 levels normalized on GAPDH values and expressed as percentage of vehicle from three independent experiments (mean ± s.e.m.). *P<0.05,**P<0.01, P<0.001*** vs 0.005% BEO (ANOVA followed by Tukey-Kramer multiple comparisons test). (<b>B, C</b>) Effect of BafA1 pretreatment on LC3II accumulation in SH-SY5Y exposed to BEO. Cells were preincubated for 2 h with BafA1 (100 nM) and then treated with BEO 0.01% (<b>B</b>) or 0.02% (<b>C</b>) for 1 h. LC3II levels were detected by western blotting. Histogram in (<b>C</b>) shows the results of densitometric analysis of LC3II relative to internal control and reported as mean ± s.e.m of three independent experiments. **P<0.01, ***P<0.001 vs vehicle treated cells, ^## P<0.01 vs BafA1 given alone (ANOVA followed by Tukey-Kramer multiple comparisons test). (<b>D</b>) Effect of autophagy inhibition by BafA1 on the percentage of viable, apoptotic and necrotic cells induced by BEO. Cells were incubated with BafA1 for 2 h and cell viability was assessed by cytofluorimetric analysis of FDA/PI stained cells 1 h after the addition of 0.02% BEO. Data are the mean ± s.e.m. of four independent experiments. *P<0.05, **P<0.01, ***P.001 vs vehicle treated cells (ANOVA followed by Tukey-Kramer multiple comparisons test).</p

BEO activates autophagy in MCF7 cells.

<p>Representative immunoblot showing LC3I to LC3II conversion and reduced p62 levels following exposure of MCF7 cells to increasing concentrations of BEO. Cells were incubated with medium containing either vehicle (ethanol, 0.009–0.027%) or BEO (0.01–0.03%) for 1 h. GAPDH was used as loading control. LC3II/GAPDH optical density (OD) ratio for the reported blot is shown. Histogram shows the results of densitometric analysis of p62 levels normalized on GAPDH values and expressed as percentage of vehicle from three independent experiments (mean ± s.e.m.). ***P<0.001 vs vehicle (Student's <i>t</i> test).</p

Fusion of autophagosomes and lysosomes in BEO-treated cells.

<p>(<b>A</b>) SH-SY5Y were treated with BEO 0.02% for 15 min, fixed and double stained with LC3 and LAMP-1 antibodies. Nuclei were counterstained with DAPI. Representative images were taken with a confocal microscopy and higher magnification views of the boxed area from the merged image are shown. White arrows indicate areas of fusion (yellow signal) between LC3 positive autophagosomes (green) and LAMP1 positive endosomes and/or lysosomes (red) indicative of a functional autophagic maturation. (<b>B</b>) Immunoblot showing the early conversion of LC3I into LC3II and the reduction of p62 following 15 min incubation with BEO. GAPDH was used as loading control. Images are representative of three independent experiments. (V =  vehicle, 0.018% ethanol).</p

Accumulation of endogenous LC3 positive structures in cells exposed to increasing concentrations of BEO.

<p>SH-SY5Y cells were exposed to vehicle (ethanol 0.018%) or BEO (0.005–0.02%) for 30 minutes and immunostained with anti-LC3 antibody; nuclei were counterstained with DAPI. Confocal analysis of endogenous LC3 intracellular distribution shows a dose-dependent increase of LC3 puncta (autophagosomes) in cells treated with BEO 0.01% and 0.02% as compared to vehicle or BEO 0.005% treated cells. The described patter of LC3 distribution was observed in three independent experiments.</p

BEO-induced autophagy is mTOR independent.

<p>(<b>A, B</b>) Treatment with BEO does not affect phosphorylation status of p70^S6K and ULK. (<b>A</b>) Representative immunoblots showing the levels of phospho p70^S6K (Thr 389; p-p70^S6K) and phospho ULK (Ser 757; p-ULK) following treatment with BEO 0.01% and 0.02% for 5 and 15 min. Total protein extracts were analyzed by western blotting for phospho p70^S6K and pULK and subsequently for total p70^S6K and total ULK. GAPDH was used as internal control. (<b>B</b>) Histograms show the results of densitometric analysis of autoradiographic bands from three independent experiments (mean ± s.e.m.) (V =  vehicle, 0.0018% ethanol). (<b>C</b>) BEO enhances autophagy induced by rapamycin. Neuroblastoma cells treated with rapamycin for 48h (3 µM) were exposed to BEO 0.02% or vehicle (Veh, 0.018% ethanol) for 1h. Protein extracts were analyzed by western blotting for phospho p70^S6K, p70^S6K, LC3 and p62. GAPDH was used as internal control. Treatment with rapamycin significantly reduced the phosphorylation status of p70^S6K and induced autophagy in SH-SY5Y cells as demonstrated by increased LC3II and reduced p62 levels; no change of p70^S6K phosphorylation accompanied autophagy induced by BEO; further enhancement of LC3 lipidation and p62 reduction was evident in rapamycin-treated cells exposed to BEO 0.02% for 1 h. Immunoblots are representative of three independent experiments.</p