Human bronchial epithelial cells, BEAS-2B, were purchased from the American Type Culture Collection (ATCC, CRL-9609) and cultured in bronchial epithelial cell growth medium (BEGM, Lonza) at 37 °C and 5% CO2. Airway Epithelial Cell Growth Medium (Promocell, C-21060) was used in small interfering RNA (siRNA) transfection experiments.
Reagents and antibodies
The sources of reagents and antibodies used in this study are as follows: IPTG was from Duchefa (Haarlem, The Netherlands); Ampicillin sodium salt and chloramphenicol were from USB (OH, USA); Penicillin–Streptomycin was from Thermo Fisher Scientific (UT, USA). MAPK Inhibitors PD98059, SP600125, SB203580 and BAY11-7082 were from Calbiochem (CA, USA). The oligonucleotides used in these experiments were synthesized by Bioneer (Seoul, Korea). Antibodies against phospho-p44/p42 MAPK (Thr202/Tyr204), p44/p42 MAPK, phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-MAPKAPK-2 (Thr222), MAPKAPK-2, phospho-IκBα (Ser32/36), IκBα, Lamin A/C and β-actin were from Cell Signaling Technology (MA, USA); GAPDH antibody was from AbFrontier (Seoul, Korea); NF-κB (p65) antibody was from Enzo Life Sciences (NY, USA).
Recombinant protein production
We prepared the recombinant proteins as previously described . Briefly, pRSET A/Del-N11 TCTP was transformed to E. coli strain BL21(DE3)pLysS and cells were grown at 37 °C and 220 rpm in Luria–Bertani medium containing the 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. The pre-culture medium was diluted 1:100 with 400 ml and cultured until OD600 reached 0.6–0.8 (Hitachi, U-3000). After IPTG was added to a final concentration of 0.4 mM, the culture was incubated at 37 °C and 200 rpm for 2 h 30 min. Cells were harvested by centrifugation at 7140×g (Sorvall, SLA-3000 rotor) for 10 min at 4 °C and stored at − 70 °C or used directly. Cell pellets were resuspend in ice-cold equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole; pH 7.4) with 1 mM PMSF and disrupted by sonication (Kyung Ill, KTA-400). Sonication was performed three times on ice for 30 s at 1/10 of maximal amplitude followed by centrifugation for 40 min at 12,000×g (Sorvall, SS34-rotor).
The supernatants containing soluble proteins were purified with HisPur™ Cobalt Resin (Thermo, 89965) according to the manufacturer’s instruction. Subsequently, the proteins were eluted and the mixtures desalted using a PD-10 desalting column (GE Healthcare, 17-0851-01) and loaded onto HiTrap Q HP column (GE Healthcare, 17-1153-01) which was equilibrated with buffer A (20 mM Tris, 1 mM EDTA, 50 mM NaCl; pH 7.4). Proteins were separated by AKTA FPLC systems (GE Healthcare) and eluted with buffer B (20 mM Tris, 1 mM EDTA, 1 M NaCl; pH 7.4) with a constant flow rate of 1 ml/min. The eluted fractions were separated by SDS-PAGE and the samples containing dTCTP were collected and desalted with PBS. Purified dTCTP was concentrated using Vivaspin 500 (Sartorius, VS0122) and stored at − 70 °C until use.
Measurement of IL-8
BEAS-2B cells were seeded at 4000 cells per well in 48-well plates (Nunc). When the cells became 60% confluent, they were washed twice with 1% penicillin–streptomycin/BEBM, and treated with or without inhibitor for 30 min at indicated concentrations followed by 10 μg/ml dTCTP. After 16–20 h, IL-8 in the media was measured with Legend MAX™ Human IL-8 ELISA Kit (BioLegnd, 431508) according to the manufacturer’s protocol.
Immunofluorescence confocal microscopy
Sterilized cover slips were coated with poly-d-lysine (Sigma) in 24 well plates (Nunc) for 2 h 30 min and BEAS-2B cells were seeded at 35,000 cells per well. After 36–48 h, cells were serum starved for 2 h and treated with PBS or 10 μg/ml dTCTP for 1 h followed by fixing with 4% paraformaldehyde (Sigma, P6148) in PBS for 15 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min on ice and blocked with 1% bovine serum albumin in permeabilization buffer for 1 h at room temperature. Rabbit anti-NF-κB (p65) antibody (Enzo, ALX-210-574) was diluted 1:300 with blocking buffer and treated to the cells overnight at 4 °C. The stained cells were washed with PBS and probed with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A11008) diluted 1:1000 in blocking buffer for 30 min at room temperature in a shaded chamber. After washing with PBS, cells were counterstained with DAPI using ProLong™ Gold Antifade Mountant (Invitrogen, P36931). Confocal microscopy was performed using a Carl Zeiss Laser Scanning Systems LSM 510.
BEAS-2B cells were washed with PBS 3 times and harvested by scraping with ice-cold lysis buffer (50 mM Tris–HCl; pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 1% Triton X-100) containing protease inhibitor cocktail (Roche, 11 836 170 001) and phosphatase inhibitor cocktail (Sigma Aldrich, P5726 and P0044). Cells were vortexed thoroughly for 30 s and set on ice for every 10 min. After 30 min, lysates were centrifuged at 12,000×g for 20 min at 4 °C, and the supernatants were collected. Protein contents were quantified by Bradford assay (Bio-Rad, CA, USA) and 5–15 μg of each sample were separated by SDS-PAGE, transferred to PVDF membranes and probed with primary antibodies followed by incubation with HRP-conjugated goat anti-mouse/rabbit IgG antibody (Bio-rad, CA, USA). Proteins of interest were visualized with chemiluminescent sensitive plus HRP substrate (Surmodics, LERI-0110-2C) and detected with LAS-3000 (Fujifilm, Tokyo, Japan).
Total RNA was isolated from cells using RNeasy Mini Kit (Qiagen, 74104) as described by the manufacturer. The isolated RNA had an A260/A280 ratio of 2.0–2.1 and 1 μg of each RNA samples were reverse transcribed into cDNA using High capacity cDNA Reverse transcription Kit (Applied Biosystems, 4368814). 1 μl of the resulting cDNA, 10 pmol of each forward and reverse primer, and the DNA polymerase mixture provided in AccuPower PCR PreMix (Bioneer, K-2016) in 20 μl reaction volume were amplified using PCR. The primers were synthesized by Genotech Co. Ltd. (Korea) and the sequences were: IL-8, 5′-CATGACTTCCA AGCTGGCCGTG-3′ (forward) and 5′-TCACTGATTCTTGGATACCACA GAG-3′ (reverse) and β-actin, 5′-CAGCTCGTAGCTCTTCTCCA-3′ (forward) and 5′-CAGCTCGTAGCTCTT CTCCA-3′ (reverse). The reaction mixtures were pre-denatured by incubating at 94 °C for 5 min followed by 27 cycles (for IL-8) or 30 cycles (for β-actin) of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and for final extension, they were incubated at 72 °C for 10 min. 8 μl of the amplified products were resolved on a 1% agarose gel containing SYBR™ Safe DNA gel stain (Invitrogen). Images were taken using E-Graph AE-9000 (Atto, Japan).
Real-time quantitative PCR
Changes in IL-8 transcripts were confirmed by real-time PCR in cells treated with various doses of dTCTP. Levels of IL-8 mRNA was analyzed using TaqMan Gene Expression Assays (Applied Biosystems, 4331182) (Assay IDs: IL-8, Hs00174103_m1; GAPDH, Hs99999905_m1) according to the manufacturer’s protocol. 2 μl of cDNA was used for 20 μl reaction, and each samples were run in triplicates. Reaction mixtures were amplified with an initial denature step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min using Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, ABI 7300). The expression of IL-8 mRNA was normalized to GAPDH mRNA. The efficiencies of the reactions were calculated by a fivefold serial dilution and estimated to be 90–95%.
BEAS-2B cells were plated at 6 × 104 cells/well into 96 well culture plate (Nunc). After 48 h at 70% confluency, BEAS-2B cells were cotransfected with 1 μg of experimental reporter plasmid along with 0.1 μg of control plasmid linked to the Renilla luciferase vector (pRL-TK, Promega) by using Lipofectamine 2000 reagent (Invitrogen). Experimental reporter vectors regulated by a synthetic promoter containing direct repeats of consensus sequences for AP-1 and NF-κB designated as pAP-1-Luc and pNF-κB-Luc were purchased from Stratagene. IL-8 promoter reporter plasmids with and without a mutation of NF-κB (GGATTTCCT to TAACTTTCCT) and AP-1 (TGACTCA to TATCTCA) binding site cloned into the pGL3-Basic vector, designated as pIL8, pIL8mutNF-κB, and pIL8mutAP-1 were kindly provided by HY Lee (Yonsei University, Korea) . At 24 h after transfection, cells were washed and stimulated with dTCTP for indicated times and stored at − 70 °C. The luciferase activity of firefly and Renilla luciferase were measured using Dual-Luciferase Reporter Assay System (Promega, E1910) and read by a microplate luminometer (Berthold Technologies, MicroLumat Plus). The frozen cells were lysed with 30 μl of passive lysis buffer and gently rocked at room temperature until complete lysis was confirmed by microscopy. After transferring 20 μl of the cell lysate to a 96-well white luminometer plate, the activities of firefly and Renilla luciferases were measured sequentially form a single sample. The results were presented as the relative ratio of experimental vector to control vector.
Electrophoretic mobility gel shift assay (EMSA)
BEAS-2B cells grown in 100 mm culture dish (Nunc) were serum starved for 2 h and stimulated with either PBS or dTCTP. The nuclear extracts were prepared using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo, 78833) according to the manufacturer’s manual. The protein contents of nuclear extracts were quantified, aliquoted, and used directly or stored at − 70 °C until use.
The sequences of the sense-strand oligonucleotides used in these experiments were as follows: NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′, AP-1, 5′-CGCTTGATGAGT CAGCCGGAA-3′ (Promega Corporation, USA), mutant NF-κB, 5′-AGTTGAGGTAA CTTTCCCAGGC-3′, and mutant AP-1, 5′-CGCTTGATATGTCAGCCGGAA-3′. The complementary pairs of oligonucleotides were annealed using a heating block. The pair of nucleotides were mixed at a molar ratio 1:1, incubated at 95 °C for 5 min, and allowed to stand overnight at room temperature.
EMSA was performed as previously described with some modification. Briefly, 6% polyacrylamide gel was pre-run for 40 min in 0.5× TBE buffer. During this time, binding reactions were performed by adding 5 μg of nuclear extracts (as 2–4 μl of NE-PER) in 5× binding buffer (20% Glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris, 2.5 mg/ml poly dI·dC). Then 3′ end-biotinylated double stranded DNA was added at a final concentration of 1 pmol and incubated at room temperature for 20 min. The specificity of the binding reaction was confirmed by pre-incubating the nuclear extracts with tenfold molar excess of unlabeled DNA. The reaction mixtures were electrophoresed and transferred to nylon membrane (Roche, 11 209 299 001) at 4 °C. The membrane was cross-linked for 10 min using E-Graph AE-9000 (Atto Incorporation, Japan) equipped with 312 nm UV transilluminator. The biotin-labeled DNA on the membrane was detected using LightShift Chemiluminescent EMSA Kit (Thermo, 20148) according to the instructions.
Small interfering RNA (siRNA)
BEAS-2B cells were seeded in 12 well plates (160,000 cells/well) and incubated for 24–36 h in growth media. At 60–65% confluency, cells were transfected with AccuTarget™ FAM labeled for Negative Control (Bioneer, SN-1021) or siRNA targeting p65 mRNA (Bioneer, 1128171) using Lipofectamine® RNAiMAX Reagent (Invitrogen) at a final concentration of 20 nM in Opti-MEM® (Thermo). After 6 h, media were changed with growth media and incubated for another 18 h. 24 h post-transfection, the transfection efficiency was confirmed by fluorescence microscopy (data not shown) and the cells were treated with or without 10 μg/ml of dTCTP for 16–20 h. The resulting supernatants were harvested and analyzed for IL-8 contents.
Data are presented as means ± standard errors. Data were analyzed with GraphPad Prisms 5 software (GraphPad Software Inc., CA, USA). Statistical significance was determined using Student’s two-tailed unpaired t test for comparisons between two groups. For more than 3 groups, one-way ANOVA analysis was performed.