Cell lines, culture, and reagents-
SH-SY5Y cell line was obtained from NCCS, Pune. All the cells were cultured in DMEM-F12 (CELL clone) with 10% FBS (Gibco) and 1% Antibiotic cocktail (CELL clone). They were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were maintained at the log phase. As the cells became confluent, they were split after treatment with Trypsin-EDTA. 2′,7′-Dichlorofluorescin diacetate (DCFDA) (#D6883) and LiCl (#L4408) from Sigma-Aldrich, Mito-tracker, (Invitrogen™ M7512), were purchased for immunofluorescence studies, LC3B Antibody (Cell Signaling, #2775), GSK-3β Antibody (Cell Signaling, #9315), a-Synuclein (cell signaling, #2642), Bcl-2Antibody (Cell Signaling, # 4223) AKT Antibody (Cell Signaling, # 4685) b-catenin Antibody (Cell Signaling, #8480) GAPDH Antibody (Cell Signaling, #5174) were used. For Western blots, Rotenone was given 24 hrs. Before assessing the cell damage. For MTT assay, Reactive oxygen species, & Mitochondrial membrane potential (MMP) detection, apoptosis analysis. LiCl was initially dissolved in dH2O before adding to DMEM. The total duration of LiCl treatment is 48hrs in which cells were pre-treated with LiCl for 24 hours and then with rotenone for another 24hr.
MTT assay and cellular morphology observation -
we first studied the cytotoxic effect of the drugs. Ten thousand cells per well (10*103 cells/well) were seeded in 96 well plates after incubation of 24 hrs. Cells were treated dose-dependently with 1mM to 100mM LiCl for 48hrs and with rotenone 25nM to 1micro M for 24 hrs. Cytotoxicity was measured as per the manufacturer’s protocol. After the treatment, MTT was added 20m lit (5mg/ml) to the wells, then incubated in a humidified incubator at 37 °C for two hrs. To allow the formation of purple formazan crystal. These crystals dissolved in 100 microliters of DMSO, and absorption was taken at 570 nm.
MMP, ROS, assessments-
SH-SY5Y Cells were harvested and re-suspended in PBS, then immediately stained with Mito-tracker (stock 1mM solution in DMSO to working 500mM) for Mitochondrial membrane potential ROS DCFH-DA (10 M, Invitrogen), and incubated at 37 °C for 30 min in darkness. After washing twice with ice-cold PBS, the samples were subject to a FAC Scan Flow Cytometer (BD). Data were analyzed by FSC express version 3.0 (De Novo Software, Los Angeles, CA, USA)
The rate of cell death was analyzed by flow cytometry following annexin5-FITC/propidium iodide (Invitrogen molecular prob, USA). According to this method, cells were seeded in 6 well plates at semi confluent stage cells were treated with LiCl (10mM,30mM) for 48 hrs followed by rotenone 100nM for 24 hrs. Cells were harvested by 0.25% trypsin and washed with PBS; then, cells were treated with FITC conjugated Annexin and incubated at 37oc for 15 min in darkness, add 2ml 1x binding buffer centrifuge at 6000 rpm for 5 min at room temp. resuspend the cell in 200ul in buffer add 5 ul PI 5-15 min on ice data were analyzed by BD-FACS Caliber instrument ( BD- biosciences, USA )
1000 cells were seeded on sterilized coverslips in 6 well plates (NEST, Thermo Fisher Scientific). After the drug treatment at standard conditions, the coverslip wells were washed with PBS for Mito-tracker. Dilute 1mM Mito-Tracker® stock solution to the final working concentration of 100nM, then remove the media from the dish and add prewarmed (37°C) staining solution containing Mito-Tracker® probe and incubate for 15 min under growth conditions. After staining, wash the cells in a fresh, pre-warmed buffer or growth medium. Carefully remove the medium/buffer covering the cells, and replace freshly prepared, pre-warmed buffer or growth medium containing 2–4% formaldehyde in complete growth medium at 37°C for 15 minutes at four °C. Cells were counterstained with DAPI after rinsing with one × PBS. The coverslips were mounted with DABCO (Sigma) over pre-cleaned slides, observed using fluorescence Microscopy fluorescence measured using ImageJ software, and statistical analysis was done using GraphPad Prism 9 software.
Western Blot –
cells treated with rotenone alone and combine with LiCl (10mM, 30mM) were collected after 48 hrs by washes with PBS. Cell lysis was done with ice-cold RIPA buffer (Sigma Aldrich, USA). The cell lysate was kept for 20 min followed by gentle vortexing in between, then centrifugation was done on 12000rpm for 10 min at 4oC; the supernatant was collected in a fresh Eppendorf tube and sorted at -80oC for further use equal quantity 50mg was used for western blot analysis protein was resolved electrophoretically on 10% SDS-PAGE gel then transferred on to PVDF membrane (Millipore, USA). PVDF membrane was blocked with 5% non-fat milk dissolved in TBST containing 0.5% tween 20. after blocking, the membrane was incubated with primary antibody GSK-3beta, AKT, Bcl-2, and LC-3B, beta-catenin, and GAPDH antibodies overnight at 4oC; the membrane was further incubated with their respective secondary antibody conjugated to ALP for 3 hrs after that membrane was exposed to BCIP-NBT solution (America USA). The relative intensity of bands was determined densitometrically by using the Quantity One software (Quantity One, Hercules, CA, USA). All data from independent experiments were expressed as the ratio to optical density values of the corresponding controls for statistical analyses.
Numerical data was represented graphically as mean ± standard deviation (SD). The student’s t-test (paired for cell lines) was applied to compare the control-treatment groups using GraphPad Prism 9 software (La Jolla, USA). P≤0.05 was considered statistically significant and was two-sided.