Cell culture and stimulation
Primary bronchial epithelial cells (PBEC) were isolated from resected lung tissue obtained from patients undergoing surgery for lung cancer as described previously (van Wetering et al. 2000). Briefly, the cells were cultured in a 1:1 mixture of DMEM (Invitrogen, Carlbad, CA, USA) and BEGM (Clonetics, San Diego, CA, USA), supplemented with 0.4% w/v BPE, 0.5 ng/ml EGF, 5 μg/ml insulin, 0.1 ng/ml retinoic acid, 10 μg/ml transferrin, 1 μM hydrocortisone, 6.5 ng/ml T3, 0.5 μg/ml epinephrine (all from Clonetics), 1.5 μg/ml BSA (Sigma, St Louis, MO, USA), 1 mM Hepes (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Cambrex, East Rutherford, NJ, USA).
Immortalized human renal PTEC (HK-2, kindly provided by M. Ryan, University College Dublin, Dublin, Ireland) were grown in serum-free DMEM/HAM-F12 (Bio-Whittaker, Walkersville, MD) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen, Breda, The Netherlands), insulin (5 μg/ml), transferrin (5 μg/ml), selenium (5 ng/ml), triiodothyronine (40 ng/ml), epidermal growth factor (10 ng/ml), and hydrocortisone (36 ng/ml, all purchased from Sigma, Zwijndrecht, The Netherlands).
Cells from the A549 human lung carcinoma cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured in RPMI 1640 medium (Gibco, Grand Island, NY), supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex, East Rutherford, NJ, USA) and 10% (v/v) heat-inactivated FCS (Gibco) at 37°C in a 5% CO2-humidified atmosphere.
ER stress was induced in epithelial cells by exposure to thapsigargin or tunicamycin. After reaching near-confluence, PBEC were exposed to thapsigargin (50 nM, Sigma) for various time periods. For the dose–response experiment PBEC from 3 different donors were stimulated with various concentrations of thapsigargin or tunicamycin (Sigma) for 6 h. Dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) served as a solvent control for both thapsigargin and tunicamycin. The dose–response experiments were repeated on HK-2 cells and A549 cells with 2 h stimulation instead of 6 h. This shorter duration of exposure in HK-2 and A549 cells was based on pilot experiments using these cell lines.
Total RNA isolation and reverse-transcription
After stimulation the cells were washed twice with PBS and total mRNA was isolated using the RNeasy mini kit (Qiagen, Valencia CA, USA). DNase I amplification grade (Invitrogen) was used to remove genomic DNA. Total RNA concentration and purity were measured on a NanoDrop spectrophotometer (NanoDrop technologies, Wilmington USA). Next, cDNA synthesis was performed with M-MLV Reverse Transcriptase (Promega, Madison WI, USA).
To amplify the spliced and unspliced XBP1 mRNA, XBP1 primers were used as described previously (Yoshida et al. 2001). PCR products were electrophoresed on 2.5% agarose gel. GAPDH (forward 5′GGATGATGTTCTGGAGAGCC3′, reverse 5′CATCACCATCTTCCAGGAGC3′) was used as a loading control. The size difference between the spliced and the unspliced XBP1 is 26 nucleotides.
Primers were designed to span the 26 base pair intron that is removed by IRE1 to obtain the spliced XBP1 mRNA (XBP1spl) (forward 5′TGCTGAGTCCGCAGCAGGTG3′ and reverse 5′GCTGGCAGGCTCTGGGGAAG3′). Also specific primers for CHOP (forward 5′GCACCTCCCAGAGCCCTCACTCTCC3′ and reverse 5′GTCTACTCCAAGCCTTCCCCCTGCG3′) and BiP (Hirota et al. 2006) were used. Quantitative PCR was carried out at 95°C for an initial 3 min followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 15 s and extension at 72°C for 30 s using IQ SYBRGreen supermix (Bio-Rad, Hercules, CA, USA). Each assay was run on a Bio-Rad CFX Real-time PCR system in triplicates and arbitrary mRNA concentrations were calculated by the Bio-Rad software, using the relative standard curve method. Stable housekeeping genes were selected using the Genorm software (Vandesompele et al. 2002). Relative mRNA concentrations of ATP5B and RPL13A (GeNorm, Primerdesign, Southampton, UK) were used as reference genes to calculate the normalized expression of the XBP1spl mRNA. The identity of the PCR products obtained with the XBP1spl primers was verified by DNA sequencing.
The results of the dose–response experiment were expressed as mean±SEM. The correlation coefficient was determined by the use of Pearson’s correlation statistics. The correlation coefficient was considered significant at p values < 0.05.