Re (Becton Dickinson Biosciences).Apoptosis inductionApoptosis was induced by exposing cells to the lysosomotropic detergent MSDH (10?5 mM; kindly provided by Gene M. Dubowchik, Bristol-Myers Squibb, Wallingford, CT, USA), glucose oxidase (GO; 1.6 mg/ml, Sigma-Aldrich) or H2O2 (570?760 mM; Sigma-Aldrich). The concentrations were optimized to induce apoptosis without necrotic contamination, as judged by morphologic examination of cell cultures. MSDH was added in serum-free medium for 24 h. All drugs (U18666A, quinacrine, 25HC, MbCD and myriocin) were omitted during the exposure. Cells were exposed to H2O2 in serum-free medium for 2 h and then purchase 520-26-3 incubated for 24 h in complete medium before analysis. GO, an enzyme that catalyzes the oxidation of glucose and generates H2O2, was freshly prepared prior to each experiment (1 mg/ml in 50 mM sodium acetate, pH 5.1). Neurons were exposed to GO in complete medium for 1 h, and then the medium was exchanged to serum free medium for 24 h.Western blot analysisProtein separation was performed as described previously [20]. Proteins were blotted onto a nitrocellulose membrane using an iBlot Dry Blotting System (Invitrogen). The following primary antibodies were used: mouse anti-LAMP-2 (1:1000; Southern Biotech, Birmingham, AL, USA) and mouse anti-glyceraldehyde3-phosphate dehydro-genase (GAPDH; 1:5000; Novus Biologicals, Littleton, Co, USA).Determination of lysosomal membrane stabilityTo analyze the integrity of lysosomes, photo-oxidation of AO (Gurr, Poole, UK) was employed as described earlier [23]. AO is a metachromatic dye that, when excited by blue light, emits red fluorescence when highly concentrated inside lysosomes and green fluorescence when diluted in the cytosol [26]. Cells seeded on coverslips were incubated with AO (2 mg/ml) for 15 min at 37uC, washed with phosphate buffered saline (PBS), and placed on the stand of a Nikon Eclipse E600 laser scanning confocal microscope. AO was excited using a 488 nm light from a 100-mW diode laser, and loss of lysosomal proton gradient was followed by capturing laser scanning micrographs every 330 ms in a ASP-015K site channel defined by bandpass filters for 495?55 nm. Green fluorescence intensity in pre-defined areas was subsequently analyzed using Volocity (PerkinElmer, Waltham, MA, USA) and plotted. The loss of lysosomal integrity was determined as the lag time from the start of blue laser irradiation until the rupture of lysosomes induced an increase of green fluorescence in the cytosol (Figure 3E).Viability analysisAfter treatment, cell cultures were morphologically examined in a phase contrast microscope and viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Calbiochem, San Diego, CA, USA) reduction assay. Cells were incubated with 0.25 mg/ml MTT for 2h at 37uC. The MTT solution was then removed and the formazan product dissolved in DMSO. The absorbance was measured at 550 nm. In addition, the amount of surviving and thus attached cells was determined using crystal violet staining. Cells were fixed in 4 paraformaldehyde for 20 min, followed by 0.04 crystal violet staining for 20 min at room temperature. The plates were washed thoroughly by dipping in H2O and subsequently air-dried. Samples were then solubilized in 1 Sodium dodecyl sulfate (SDS) before absorbance was measured at 550 nm. Caspase-3-like activity was analyzed using the substrate Ac-DEVD-AMC (Becton, Dickinson and Company, Franklin Lakes, NJ) according.Re (Becton Dickinson Biosciences).Apoptosis inductionApoptosis was induced by exposing cells to the lysosomotropic detergent MSDH (10?5 mM; kindly provided by Gene M. Dubowchik, Bristol-Myers Squibb, Wallingford, CT, USA), glucose oxidase (GO; 1.6 mg/ml, Sigma-Aldrich) or H2O2 (570?760 mM; Sigma-Aldrich). The concentrations were optimized to induce apoptosis without necrotic contamination, as judged by morphologic examination of cell cultures. MSDH was added in serum-free medium for 24 h. All drugs (U18666A, quinacrine, 25HC, MbCD and myriocin) were omitted during the exposure. Cells were exposed to H2O2 in serum-free medium for 2 h and then incubated for 24 h in complete medium before analysis. GO, an enzyme that catalyzes the oxidation of glucose and generates H2O2, was freshly prepared prior to each experiment (1 mg/ml in 50 mM sodium acetate, pH 5.1). Neurons were exposed to GO in complete medium for 1 h, and then the medium was exchanged to serum free medium for 24 h.Western blot analysisProtein separation was performed as described previously [20]. Proteins were blotted onto a nitrocellulose membrane using an iBlot Dry Blotting System (Invitrogen). The following primary antibodies were used: mouse anti-LAMP-2 (1:1000; Southern Biotech, Birmingham, AL, USA) and mouse anti-glyceraldehyde3-phosphate dehydro-genase (GAPDH; 1:5000; Novus Biologicals, Littleton, Co, USA).Determination of lysosomal membrane stabilityTo analyze the integrity of lysosomes, photo-oxidation of AO (Gurr, Poole, UK) was employed as described earlier [23]. AO is a metachromatic dye that, when excited by blue light, emits red fluorescence when highly concentrated inside lysosomes and green fluorescence when diluted in the cytosol [26]. Cells seeded on coverslips were incubated with AO (2 mg/ml) for 15 min at 37uC, washed with phosphate buffered saline (PBS), and placed on the stand of a Nikon Eclipse E600 laser scanning confocal microscope. AO was excited using a 488 nm light from a 100-mW diode laser, and loss of lysosomal proton gradient was followed by capturing laser scanning micrographs every 330 ms in a channel defined by bandpass filters for 495?55 nm. Green fluorescence intensity in pre-defined areas was subsequently analyzed using Volocity (PerkinElmer, Waltham, MA, USA) and plotted. The loss of lysosomal integrity was determined as the lag time from the start of blue laser irradiation until the rupture of lysosomes induced an increase of green fluorescence in the cytosol (Figure 3E).Viability analysisAfter treatment, cell cultures were morphologically examined in a phase contrast microscope and viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Calbiochem, San Diego, CA, USA) reduction assay. Cells were incubated with 0.25 mg/ml MTT for 2h at 37uC. The MTT solution was then removed and the formazan product dissolved in DMSO. The absorbance was measured at 550 nm. In addition, the amount of surviving and thus attached cells was determined using crystal violet staining. Cells were fixed in 4 paraformaldehyde for 20 min, followed by 0.04 crystal violet staining for 20 min at room temperature. The plates were washed thoroughly by dipping in H2O and subsequently air-dried. Samples were then solubilized in 1 Sodium dodecyl sulfate (SDS) before absorbance was measured at 550 nm. Caspase-3-like activity was analyzed using the substrate Ac-DEVD-AMC (Becton, Dickinson and Company, Franklin Lakes, NJ) according.
