Ng of wt and LAMPnull MEFs (scale bar 10 mm). B) Phase

Ng of wt and LAMPnull MEFs (scale bar 10 mm). B) Phase contrast images (scale bar 5 mm) and C) viability (n = 4) of wt and LAMPnull MEFs 24 h after H2O2 exposure. Viability was measured by crystal Calciferol site violet staining and expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05, ns; non-significant. doi:10.1371/journal.pone.0050262.greplaced with fresh media (10 ng/ml b-FGF). Cells were used for experiments at day 12?8. LAMP-12/2, LAMP-22/2, LAMPnull and wt mouse embryonic fibroblasts (MEFs) were generated as previously described [30]. The cells were grown in Dulbecco’s minimum essential medium containing 50 IU/ml penicillin-G, 50 mg/ml streptomycin and 10 fetal calf serum (all from Gibco). Cells were incubated in humidified air with 5 CO2 at 37uC andwere subcultured twice a week. Prior to experiments, fibroblasts were trypsinized and seeded at a density that allowed them to reach 80 1326631 confluence at the time of apoptosis induction. Cells were pre-treated with U18666A (0.25-3 mg/ml), quinacrine (2 mM) and 25-HC (1 mg/ml; all from Sigma-Aldrich, St. Louis, MO, USA) for 48 h. MbCD (400?00 mM; Sigma-Aldrich) was added to cells for 1 h to allow endocytosis and then removed andLysosomal Stability Is Regulated by Cholesterolcells were chased for 24 h. This approach was shown to deplete cholesterol from the lysosomal membrane rather than the plasma membrane [44]. Cells were treated with myriocin (10 mM; SigmaAldrich) or vehicle (dimethyl sulfoxide; DMSO) for 48 h before analysis or apoptosis induction.Flow cytometric determination of Lysotracker fluorescenceCells were stained with 50 nM Lysotracker green-26 (Invitrogen) for 5 min at 37uC and detached by trypsinization. Lysotracker fluorescence was analyzed in a LSR flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) using a 488 nm argon laser and the resulting fluorescence was detected in the FL1 channel using a 530628 nm filter. Data from 10000 cells were collected and was analyzed using CellQuest software (Becton Dickinson Biosciences).Apoptosis Emixustat (hydrochloride) 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 l.Ng of wt and LAMPnull MEFs (scale bar 10 mm). B) Phase contrast images (scale bar 5 mm) and C) viability (n = 4) of wt and LAMPnull MEFs 24 h after H2O2 exposure. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05, ns; non-significant. doi:10.1371/journal.pone.0050262.greplaced with fresh media (10 ng/ml b-FGF). Cells were used for experiments at day 12?8. LAMP-12/2, LAMP-22/2, LAMPnull and wt mouse embryonic fibroblasts (MEFs) were generated as previously described [30]. The cells were grown in Dulbecco’s minimum essential medium containing 50 IU/ml penicillin-G, 50 mg/ml streptomycin and 10 fetal calf serum (all from Gibco). Cells were incubated in humidified air with 5 CO2 at 37uC andwere subcultured twice a week. Prior to experiments, fibroblasts were trypsinized and seeded at a density that allowed them to reach 80 1326631 confluence at the time of apoptosis induction. Cells were pre-treated with U18666A (0.25-3 mg/ml), quinacrine (2 mM) and 25-HC (1 mg/ml; all from Sigma-Aldrich, St. Louis, MO, USA) for 48 h. MbCD (400?00 mM; Sigma-Aldrich) was added to cells for 1 h to allow endocytosis and then removed andLysosomal Stability Is Regulated by Cholesterolcells were chased for 24 h. This approach was shown to deplete cholesterol from the lysosomal membrane rather than the plasma membrane [44]. Cells were treated with myriocin (10 mM; SigmaAldrich) or vehicle (dimethyl sulfoxide; DMSO) for 48 h before analysis or apoptosis induction.Flow cytometric determination of Lysotracker fluorescenceCells were stained with 50 nM Lysotracker green-26 (Invitrogen) for 5 min at 37uC and detached by trypsinization. Lysotracker fluorescence was analyzed in a LSR flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) using a 488 nm argon laser and the resulting fluorescence was detected in the FL1 channel using a 530628 nm filter. Data from 10000 cells were collected and was analyzed using CellQuest software (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 l.