NaIO3, N-acetyl cysteine (NAC), Nec-1, dichlorodihydrofluorescein diacetate (H2DCFDA), MitoSOX, propidium iodide (PI), oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, antimycin A, bafilomycin A1 (Baf A1), U0126, SP600125, SB203580, Akt inhibitor (AktI), 3-methyladenine (3-MA), and Trolox were obtained from Sigma-Aldrich Co (St Louis, MO, USA). The antibodies specific for phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-JNK (T183/Y185), JNK, phospho-p38 (T180/Y182), p38, phospho-dynamin-related protein (DRP)-1 (S616), DRP-1, poly(ADP-ribose) polymerase 1 (PARP1), γ-H2AX, LC3, p62, and Tom 20 were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody specific for β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for mitofusin (MFN)-1, MFN-2, optic atrophy 1 (OPA-1), phospho-Akt (T308) and Akt were purchased from Abcam (Cambridge, UK). Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12), trypsin-EDTA, penicillin, ampicillin and streptomycin were from Invitrogen (Rockville, MD, USA). The ECL reagent (Western blotting lightening chemiluminescence reagent plus) was purchased from PerkinElmer (Wellesley, MA, USA).
Adult human RPE cell line ARPE-19 was purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). These cells were maintained in DMEM/F12 supplemented with 10% fetal calf serum (GibcoBRL, Invitrogen Life Technologies, Carlsbad, CA, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich Co.). The cells were cultured in a humidified incubator at 37 °C and 5% CO2. For most of the experiments, cells reaching a 90–95% of confluence were starved and synchronized in serum-free DMEM for 24 h before they were subjected to further analysis.
Annexin V-FITC/PI assay
The cell surface exposure of phosphatidylserine and the plasma membrane impairment of cells were assessed using Annexin V-FITC Apoptosis Detection Kit (Calbiochem). Briefly, suspension of treated/control ARPE-19 cells, containing 5 × 105 cells, was washed with PBS and re-suspended in 0.5 ml cold binding buffer. Then, 1.25 μl of Annexin V-FITC was added and the cells were incubated in the dark for 15 min at room temperature. Following incubation, the cells were centrifuged at 100×g for 5 min and the supernatant was removed. The cell pellet was re-suspended in 0.5 ml cold binding buffer, and 10 μl of the 30 μg/ml propidium iodide (PI) solution was added. Cell samples were placed on ice, away from light, and FITC and PI fluorescence were immediately measured by using flow cytometer (Cytomics FC500; Beckman-Coulter, Brea, CA, USA). Data were analyzed using Cell Quest Pro software (Becton Dickinson, Franklin Lakes, NJ, USA). The populations of live cells, early apoptotic cells, late apoptotic and necrotic cells were determined.
Determination of the cytosolic ROS and mitochondrial ROS
Cytosolic ROS production was detected using H2DCFDA for H2O2 and mitochondrial ROS was detected using mitoSOX. After drug treatment, ARPE-19 cells were washed with PBS and incubated with 10 μM H2DCFDA or 5 μM MitoSOX Red at 37 °C for 30 min. Subsequently, the cells were washed in PBS, trypsinized and the fluorescence intensity was measured by flow cytometry (Cytomics FC500; Beckman-Coulter) at excitation/emission wavelengths of 485/530 nm and 510/580 nm for DCFDA and mitoSOX, respectively. For each sample, ROS production was expressed as mean fluorescence ratio (fluorescence of exposed cells/fluorescence of control cells) from the same experiment.
Cell lysate preparation and Western blot analysis
After stimulation, the medium was aspirated. Cells were rinsed twice with ice-cold PBS, and 25–100 μl of cell lysis buffer (20 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Triton X-100, 1 mM MgCl2, 25 mM β-glycerophosphate, 50 mM NaF, 100 μM Na3VO4, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin) was then added to each well. After harvesting, cell lysates were sonicated and centrifuged, and equal protein amounts of soluble protein, as determined by the Bradford protein assay, were denatured, subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane. Non-specific binding was blocked with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.02% Tween 20) containing 5% non-fat milk for 1 h at room temperature. After immunoblotting with the first specific antibody, membranes were washed three times with TBST and incubated with a horseradish peroxidase (HRP) conjugated secondary antibody for 1 h. The dilution folds of first specific antibodies and β-actin were 1:1000 and 1:10,000, respectively. After three washes with TBST, the protein bands were detected with enhanced chemiluminescence detection reagent. To make sure equal amounts of sample protein applied for electrophoresis and immunoblotting, β-actin was used as an internal control.
Measurement of mitochondrial oxygen consumption rate
The oxygen consumption rate (OCR) was measured by the extracellular flux analyzer XF24 (Seahorse Bioscience, Houston, TX, USA) as we previously described . Cells were plated at 4 × 105 cells/well in a Seahorse 24-well V7 microplate (Seahorse Bioscience) and cultured in complete DMEM growth medium for 24 h in a 5% CO2 incubator at 37 °C. Then, the medium was removed and cells were incubated in XF assay medium in the absence of NaHCO3 and FBS for 1 h at 37 °C in measuring chamber without CO2 input. The mitochondrial complex inhibitors (oligomycin, FCCP, antimycin A1/ rotenone) were freshly prepared in XF assay media. After 26 min of measuring the basal respiration, oligomycin (2.5 μM) was injected, followed by FCCP (1 μM) at 50 min, and antimycin A (2.5 μM) /rotenone (2.5 μM) at 74 min. OCR was recorded as pMoles per minute. ATP turnover was measured after the treatment with oligomycin. Averages of three wells were taken per data point.
Cells were initially fixed with 4% paraformaldehyde at 37 °C followed by permeabilization with 0.2% Triton X-100 for 15 min, and blocking by BSA (5%) and normal IgG (1:300) for 1 h. For mitophagy measurement, immunostaining was then performed using primary antibody against Tom20 or LC3 (Abcam, Cambridge, UK) in 1% BSA overnight at 4 °C. After washing with PBS, cells were incubated with secondary antibody in 1% BSA in PBS for 1 h at room temperature and then mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were acquired using a 100 X Plan-Neofluar oil objective of LSM 880 with Airyscan SR microscopy (Carl Zeiss Micro Imaging GmbH, Jena, Germany). The co-localization of Tom20 (marker of mitochondria) and LC3 (marker of autophagosome) was determined by Zen co-localization software and on a pixel by pixel basis. Every pixel in the image was plotted in the scatter diagram based on its intensity level from each channel. The co-localization coefficients were measured for each channel.
All data were obtained from at least three separate experiments and presented as mean ± standard error mean (S.E.M.). Analysis of variance was used to assess the statistical significance of the differences. A p value of less than 0.05 was considered statistically significant.