Fig 2: Compartmentalization of specific autophagy molecules in physiological conditions (A) and within the area penumbra neurons (B). In detail, in control conditions (A), LC3 (1), which stains the autophagosomes, co-localizes with HSP70 (2) within the same vacuoles. In ischemia (B), there is a loss of both LC3 (1) and HSP70 (2) in the vacuoles as shown by the downward arrows, while both molecules greatly increase within cytosol (as shown by triple upward arrows in B). In the lysosome compartment both LAMP1 (3) and cathepsin-D (CAT D) (4) can be found in control conditions (A). During ischemia (B), there is a loss of LAMP1 (3) both in vacuoles (double downward arrows) and cytosol (double downward arrows). In ischemia (B), cathepsin-D (4) is reduced within vacuoles (double downward arrow) while it markedly increases in the cytosol (double upward arrows). In these conditions, the co-localization of the autophagosome marker LC3 (1) with the lysosome marker LAMP1 (3) within vacuoles is suppressed. This occurs more markedly than the suppression of each molecule from autophagosome and lysosome, respectively. This suggests that the massive defect in autophagolysosome formation is also due to a decrease in merging between vacuoles (5).

Fig 3: Heat Shock Protein 70 (HSP70) + lysosomal-associated membrane protein 1 (LAMP1) co-immuno-stained vacuoles decrease within the area penumbra. (A) shows representative transmission electron microscopy (TEM) micrographs of HSP70 + LAMP1 co-immuno-stained vacuoles within a cortical neuron from a region homologous to the area penumbra from a sham-operated mouse (Sham). (B) shows HSP70 + LAMP1 co-immuno-stained vacuoles within a cortical neuron from the area penumbra from an ischemic mouse (Ischemia). Arrows point to HSP70 (20 nm) and arrowheads point to LAMP1 (10 nm) immuno-gold particles within vacuoles. The insert report HSP70 + LAMP1 immuno-gold particles within vacuoles at higher magnification. Graph (C) reports the number of HSP70 + LAMP1 immuno-gold double-stained vacuoles per cell from the penumbra arewa (Ipsi/Ischemia) and homologous regions from the contralateral side (Contra/Ischemia) and from sham-operated mice (Sham). Values are given as the mean ± S.E.M. per cell counted in 120 cells from each group. * p = 0.05 compared with other groups. Scale bar = 0.2 µm (A,B), 0.1 µm (inserts).

Fig 4: Within the area penumbra, Heat Shock Protein 70 (HSP70) co-localizes with microtubule-associated protein I/II-Light Chain 3 (LC3). (A) shows representative pictures of LC3 immunofluorescence (red), HSP70 immunofluorescence (green) and merging these antigens. Graph (B) reports LC3-positive cells within the area penumbra (Ischemia) compared with the control (Sham). Graph (C) reports HSP70-positive cells within the area penumbra (Ischemia) compared with the control (Sham). Arrows point the merging of LC3 with HSP70. Graph (D) reports LC3- + HSP70-positive cells in the ischemic area penumbra (Ischemia) compared with the control (Sham). Values are reported as percentage of control and are given as the mean ± S.E.M. of 12 values for each group (n = 3 slices × N = 4 mice). * p = 0.05 compared with other groups. Scale bars = 50 µm; 10 µm (inserts) (A).

Fig 5: Representative HSP70 immuno-staining. This picture shows Heat Shock Protein 70 positive neurons stained with primary antibodies against inducible HSP70 within the area penumbra (Ischemia, in the representative picture) compared with the homologous area from a control mouse (Sham). Arrows point to HSP70 immuno-positive neurons. Scale bar = 100 µm; Scale bar (insert) = 50 µm.