Cell grouping and treatments
Human prostate cancer DU-145 cells were provided by the Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China) and were cultured in modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Logarithmic-growth-phase DU-145 cells were collected to be applied in the experimental analysis. The cells were divided into 5 groups: Control group treated with 10 µl dimethyl sulfoxide (DMSO) into the medium for follow-up incubation; low-dose As (L-As) group, moderate-dose As (M-As) group and high-dose As (H-As) group treated with 10 µl As-DMSO solution at a concentration of 20, 50 and 100 nmol/l, respectively; and tauroursodeoxycholic acid (TUDCA) group treated with 10 µl TUDCA-DMSO solution (EMD Millipore, Billerica, MA, USA).
Flow cytometry analysis
Flow cytometry was used to analyze the apoptotic percentage of DU-145 cells. In brief, 1×106 DU-145 cells were harvested, centrifuged at 60 × g, 4°C for 5 min and resuspended in phosphate-buffered saline. Subsequently, 5 µl Annexin V (1 µg/ml; Beckman Coulter, Inc., Brea, CA, USA) was added into cells and incubated at room temperature for 15 min. Subsequently, propidium iodide (1 µg/ml) was added and incubated for 5 min at room temperature. All staining incubation steps were performed in the dark. Next, cell apoptosis was detected using a FC500 series flow cytometer and MXP software (version 2.2), and the data were analyzed by CXP_9.3 version software (both from Beckman Coulter, Inc. Brea, CA, USA).
Cell proliferation assay
Cells were seeded onto 96-well plates at a density of 1.0×104 cells/well. The culture medium was replaced with DMSO medium solution, H-As-DMSO medium solution (100 nmol/l) and TUDCA-DMSO medium solution for 24 h. Subsequently, 10 µl Cell Counting kit-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added at 12, 24, 48 and 72 h, respectively. DU-145 cells were incubated at 37°C in 5% CO2 for another 4 h. The optical density value of each well was measured at 450 nm using a microplate reader (HBS-1096A; http://www.detielab.com/).
RT-qPCR
Cells were collected from each group and the total RNA was extracted using TRIzol kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The purity and integrity of total RNA were checked and reverse transcription was performed using an Easy Script First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Co., Ltd., Beijing, China). The conditions of cDNA synthesis were as follows: 25°C, 10 min followed by 42°C, 30 min according to the manufacturer's protocol. A SYBR-Green master kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and 1 µl cDNA were used to prepare the PCR reaction system. All mRNA quantification data were normalized to β-actin using the 2-∆∆Cq method (22).
The sequences of the primers used were as follows: BiP forward, 5′-GGTATTGAAACTGTGGGAGGTG-3′ and reverse, 5′-GATTGTCTTTTGTCAGGGGTCT-3′; CHOP forward, 5′-CTGAGTCATTGCCTTTCTCCTT-3′ and reverse, 5′-CCACTTTCCTTTCATTCTCCTG-3′; caspase-12 forward, 5′-GAAGGAATCTGTGGGGTGAA-3′ and reverse, 5′-TCCCTTTGCTTGTGGGATACC-3′; and β-actin forward, 5′-TGGCACCCAGCACAATGAA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The reaction conditions were as follows: 94°C for 5 min, followed by 35, 37 or 40 cycles of 94°C for 30 sec, 53°C for 30 sec and 72°C for 30 sec. The reaction was performed using a RT-qPCR instrument (Biometra GmbH, Göttingen, Germany).
Western blot analysis
The total protein was extracted using Protein Extraction Buffer (Bioo Scientific Corporation, Austin, TX, USA) and the concentration was measured using bicinchoninic acid protein quantification kit (Pierce; Thermo Fisher Scientific, Inc.). The protein (40 µg) from the samples of each group was subjected to 10% SDS-PAGE, prior to being transferred onto polyvinylidene difluoride membranes. Next, the membranes were blocked with 5% skimmed milk for 1 h and incubated overnight at 4°C with the following primary antibodies diluted in bovine serum albumin solution (50 g/l): Rabbit anti-human IRE1 polyclonal antibody (dilution, 1:1,000; no. ab37073, Abcam, Cambridge, UK), rabbit anti-human p-PERK and PERK polyclonal antibodies (dilution, 1:500; no. sc-32577 and sc-13073, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and rabbit anti-human AFT4 and AFT6 antibodies (dilution, 1:200; no. 11815 and 65880, Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Following washing with TBST (TBS, with 1 ml/l Tween-20) 3 times (for 5 min each time), membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:10,000; ab6721; Abcam) for 1 h at room temperature. Protein expression was detected using an enhanced chemiluminescent agent (Boster Biological Technology, Pleasanton, CA, USA). Protein expression levels were normalized to GAPDH and ImageJ 2.1 software (National Institutes of Health, Bethesda, MD, USA) was used to scan and quantify the gray values.
Statistical analyses
Data are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 statistical software (IBM Corp., Armonk, NY, USA). Differences among multiple groups were assessed using one-way analysis of variance, followed by Dunnett's method. P<0.05 was considered to indicate a statistically significant difference.