Mouse neuroblastoma Neuro2a cells were purchased from the Health Science Research Resources Bank (Tokyo, Japan). The cells were maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum with 100 units/ml penicillin and 100 g/ml streptomycin (Gibco BRL, Grand Island, NY, USA). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. The culture medium was replaced every 2–3 days. To prepare cell suspensions, the cells were treated with trypsin (0.25%)-EDTA (1 mM) (Gibco BRL, Grand Island, NY, USA), transferred to a 6-cm culture plate at a density of 1 × 106 cells per dish, and cultured overnight.
Measurement of cell viability using the MTT assay
Neuro2a cells were placed at a density of 1 × 104 cells per well in a 96-well cell culture plate, and cell viability was determined with a cell proliferation kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, after exposure of the cells to 2 mM bupivacaine for a period of 1 to 12 h, 10 μl 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was added to each well, and the cells were incubated at 37°C for 4 h. The supernatants were aspirated carefully and 100 μl of solubilization buffer (10% SDS in 0.01 M HCl) was added to each well. The absorbance at 550 nm was measured with a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).
Assessment of caspase-3 activity
Caspase-3 activity in cultured Neuro2a cells was measured with a caspase-3/CPP32 fluorometric assay kit (Medical & Biological Laboratories, Nagoya, Japan) according to the manufacturer’s instructions. These assays are based on the detection of the cleavage products of a fluorometric caspase substrate for caspase-3; DEVD-AFC (Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin). In brief, cells were homogenized in the cell lysis buffer supplied in the kit. Samples (200 μg protein) were then mixed with 2× reaction buffer containing 10 mM dithiothreitol (DTT). After incubation at 37°C for 2 h, free AFC that had been cleaved from the fluorometric substrate was quantified using a Fluoroskan Ascent FL microplate fluorometer (Labsystems, Helsinki, Finland) with excitation/emission (Ex/Em) of 400/505 nm, as reported previously
Analysis of DNA ladder formation
DNA fragmentation in Neuro2a cells exposed to bupivacaine was measured with an apoptotic DNA ladder assay kit (Roche Applied Sciences) according to the manufacturer’s instructions. Briefly, 200 μl of a cell suspension in PBS was mixed with 200 μl of binding buffer supplied in the kit. After incubation for 10 min at room temperature, 100 μl of isopropanol was added to the sample and mixed by vortexing. Total genomic DNA was then isolated by using glass fiber filters, treated with RNase A (400 μg/ml) at 37°C for 1 h, electrophoresed in a 1.5% agarose gel, and visualized with ethidium bromide staining under UV light.
Measurement of ROS
The OxiSelect™ intracellular ROS assay kit (Cell Biolabs, Inc., San Diego, CA) was used according to the manufacturer’s instructions. This assay uses the cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The fluorescence intensity is proportional to the concentration of ROS within the cell cytosol. Briefly, Neuro2a cells were placed in a clear 96-well cell culture plate (5 × 104 cells per well) overnight in the incubator. The cells were then exposed to DCFH-DA in medium for 30 min. After being washed twice with PBS, the cells loaded with DCFH-DA were exposed to 2 mM bupivacaine for periods from 0.5 to 9 h. The cells were lysed by adding 100 μl of cell lysis buffer, mixing thoroughly, and incubating for 5 min at room temperature. The fluorescence was read with a fluorometric plate reader at 480/530 nm.
Quantitative real-time polymerase chain reaction (qPCR) analysis
Total RNA (1 μg) was extracted from cultured Neuro2a cells with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with the ReverTra Ace® qPCR RT kit (Toyobo, Osaka, Japan). qPCR was performed with the ABI StepOne Plus real-time PCR system and a TaqMan Gene Expression Assay (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. The primers and TaqMan MGB probe for mouse WDR35 (Mm00552650_m1) were purchased from Applied Biosystems. The amount of WDR35 PCR product was calculated relative to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Mm99999915_g1; Applied Biosystems) and was compared between experimental and control groups by the ΔΔCT method, as reported previously
Western blot analysis
Protein samples from cultured Neuro2a cells were homogenized in sample buffer [50 mM Tris–HCl, pH 6.8, 0.2 M DTT, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue (BPB)] containing a mixture of protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Applied Sciences) and heated in boiling water for 5 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). These membranes were probed with anti-WDR35 peptide antibody (amino acids 459–473, 1:500), which was designed, produced, and purified by Medical & Biological Laboratories (Nagoya, Japan), or with antibodies against p38 MAPK, phospho-p38 (P-p38) MAPK, or GAPDH (Cell Signaling Technology, Danvers, MA, USA; 1:1000). Detection was performed with the Western blotting reagent ECL Prime (GE Healthcare, Buckinghamshire, UK). Protein levels were quantified by densitometric scanning with the Gel-Pro Analyzer (Media Cybernetics, Inc., USA) and expressed as the ratio to GAPDH levels as described previously
Small interfering RNA (siRNA) transfection
VNeuro2a cells were transfected with 5 nM WDR35-specific siRNA (siRNA ID: s93029; Ambion, Austin, TX, USA) or 10 nM control siRNA (negative control #1 siRNA, catalog no. 4390843; Ambion) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Effects of WDR35 siRNA on the expression of WDR35 mRNA were tested with qPCR after 24 h of transfection. In order to examine the effect of WDR35 siRNA on bupivacaine- or H2O2-induced apoptosis, after 24 h of transfection with 5 nM WDR35 siRNA, cells were further incubated with bupivacaine (2 mM) or H2O2 (0.5 mM) for 3, 6, or 9 h, and caspase-3 activation was then examined in these cells.
All results were expressed as the mean ± standard error of the mean (SEM). Data were analyzed with one-way analysis of variance (ANOVA), and significant differences between treatments were assessed by use of Tukey’s test. Differences were considered significant at P < 0.05.