Cell lines and culture
Human esophageal carcinoma cell lines TE-1 and Eca109 were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. Human esophageal carcinoma cell lines KYSE150 and KYSE510 were kindly provided by Dr. Qian Tao from The Chinese University of Hong Kong, HongKong, China. Immortalized human keratinocyte cell line HaCaT derived from human adult trunk skin was previous described [27, 28]. TE-1, Eca109, KYSE150 and KYSE510 cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. HaCaT was cultured in DMEM medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and antibiotics as described above. All cell lines were incubated at 37˚C in a humidified atmosphere containing 5% CO2.
Chemicals and cell treatments
The specific NF-κB inhibitor Bay11-7082 (Calbiochem, Darmstadt, Germany) was prepared as a stock solution of 20 mM in DMSO (Sigma, St. Louis, MO). Subconfluent cells were treated with the compound at indicated concentrations for an indicated time. Detailed treatment procedures were described in figure legends. The final concentration of DMSO in the culture media was kept less than 0.1% which had no significant effect on the cell growth. Vehicle controls were prepared for all treatments.
The pGL2-Mcl-1-κBwt (Addgene plasmid 19132) which contains a 325 bp long human Mcl-1 promoter fragment including NF-κB binding-site (GGGGTCTTCC) and the pGL2-Mcl-1-κBmt (Addgene plasmid 19133) in which the κB site sequence GGGGTCTTCC being changed to GTTGTCTTCC were constructed by Dr. El-Deiry  and obtained through Addgene (Cambridge, MA). The pGL2-Basic vector was purchased from Promega (Madison, WI). The pGL3-Basic vector and pGL3-NF-κB-Luc were the same as described previously [29, 30]. Expression plasmid of dominant negative mutant of IκBα (pcDNA3-DNMIκBα)  and the pcDNA3.1 empty vector  were identical to those used previously. The human full-length Mcl-1 expression vector pCMV6-A-Puro-Mcl and pCMV6-A-Puro empty vector were kindly provided by Dr. Chengchao Shou .
Transfection and luciferase reporter assays
Cells were cultured in 24-well plates at a density of 1 × 105 per well overnight and transfected with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. In luciferase assay for NF-κB transactivation, each transfection contained 800 ng/well of pGL3-Basic or pGL3-NF-κB-Luc together with 40 ng/well of internal control pRL-SV40 (Promega, Madison, WI) (Total DNA 840 ng/well). 24 h after transfection, cells were either left untreated (DMSO) or treated with 20 μM Bay11-7082 for 12 h. Cells were harvested at 36 h after transfection and lysates were analyzed for luciferase activity using the Dual Luciferase Reporter assay (Promega, Madison, WI) with a GloMax™ Microplate Luminometer (Promega, Madison, WI). In luciferase assay for the Mcl-1 promoter, each transfection contained 400 ng/well of pGL2-Basic, pGL2-Mcl-1-κBwt or pGL2-Mcl-1-κBmt together with 400 ng/well of pcDNA3.1 or pcDNA3-DNMIκBα expression plasmid. Each transfection contained 40 ng/well of pRL-SV40 as internal control (Total DNA 840 ng/well). 24 h after transfection, cells were either left untreated (DMSO) or treated with 20 μM Bay11-7082 for 12 h. Cells were harvested at 36 h after transfection and lysates were analyzed as described above. The pRL-SV40 was co-transfected in all experiments to correct the variations in transfection efficiency. The data represent the mean ± S.D. of at least two independent experiments performed in triplicate.
TE-1 cells were grown in 6-well plates at a density of 3 × 105 cells per well overnight. Cells reached 60-70% confluency on the day of transfection and were transfected with a p50 (sc-29407; 100 pmol), a p65 (sc-29410; 100 pmol) or a scrambled control (sc-37007; 100 pmol) siRNA (all from Santa Cruz Biotechnology) using HiPerFect transfection reagent (Cat no: 301705, Qiagen) for 72 h according to the manufacturer’s instructions. Cells were harvested for protein extraction and immunoblotting to confirm p50 or p65 knockdown.
Cell viability assay
Cell viability assays were performed using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. The assay is based on the cleavage of WST-1 to formazan dye by cellular mitochondrial dehydrogenases. Because cleavage of WST-1 to formazan dye occurs only in viable cells, the amount of dye produced, measured in OD values, directly corresponds with the number of viable cells present in the culture. Briefly, TE-1 cells were firstly transfected with the control, p50 or p65 siRNA in six-well plates as described above. To investigate whether reintroduction of Mcl-1 restored cell viability, 24 h following the first transfection, a second transient transfection was carried out to ectopically express Mcl-1. Each transfection contained 2 μg pCMV6-A-Puro empty vector or pCMV6-A-Puro-Mcl construct using SuperFect transfection reagent (Cat no: 301305, Qiagen) according to the manufacturer’s instructions. At 24 h post-transfection, cells were trypsinized, an aliquot of cells was maintained in six-well plate, harvested at 120 h after NF-κB subunit siRNA transfection and analyzed the Mcl-1 levels by Western blotting. The remainder was transferred as six replicates to 96-well plates at a concentration of 2.5 × 103 cells per well in 100 μl of complete RPMI 1640. After culturing for another 24, 48, 72 h (i.e. 72 h, 96 h, 120 h after each siRNA transfection, respectively), 10 μl of WST-1 was added to each well and cells incubated for 2 h at 37°C. The cellular reduction of WST-1 to formazan and its absorbance were measured at 450 nm.
Protein preparation and western blotting
Cultured cells were harvested and whole cell lysates were prepared according to the method previously described . Nuclear extracts were prepared using a Nuclear Extract kit (Cat. no. 40010, Active Motif, Carlsbad, CA) following the manufacturer’s instructions. Protein concentration was determined using the BCA Assay Reagent (Cat. no. 23228, Pierce, Rockford, IL). Western blotting was performed as previously described . The following antibodies were used for immunodetection with appropriate dilutions: Mcl-1 (sc-819, 1:1000), p50 (sc-114, 1:1000), p52 (sc-298, 1:1000), p65 (sc-8008, 1:1000), c-Rel (sc-272, 1:1000), RelB (sc-226, 1:1000) and GAPDH (sc-47724, 1:2000) (all from Santa Cruz, CA); Histone H3 (#9715, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA); β-actin (A5316, 1:5000) was purchased from Sigma (St. Louis, MO).
mRNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized from 2 μg of total RNA using the Reverse Transcription System Kit (Cat. No. A3500, Promega, Madison, WI). The resulted cDNA was subjected to PCR (94°C for 5 min followed by 34 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 40 s, and an extension for 10 min at 72°C) using primers designed for human Mcl-1 : sense, 5′-cggcagtcgctggagattat-3′ and antisense, 5′-gtggtggtggttggtta-3′, yield a 573-bp product; or for GAPDH: sense, 5′-caaagttgtcatggatgacc-3′ and antisense, 5′-ccatggagaaggctgggg-3′, yield a 195-bp product. Real-time RT-PCR experiments were done in triplicate as described previously  and the primers used were as following : forward 5′-gggcaggattgtgactctcatt-3′; reverse 5′-gatgcagctttcttggtttatgg-3′. The relative Mcl-1 mRNA expression levels were calculated according to the comparative CT (∆∆CT) method after normalizing to GAPDH expression. Semiquantitive RT-PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide. The identity of Mcl-1 PCR product was confirmed by direct sequencing after purification.
Electrophoretic mobility shift assays
Nuclear proteins from cultured cells were prepared and protein concentration was determined as described above. EMSA was performed using the LightShift™ Chemiluminescent EMSA Kit (Cat. No. 20148, Pierce, Rockford, IL) following the manufacturer’s instructions. The reaction mixtures (20 μl) containing 8 μg nuclear extracts were incubated with 2 nM of biotin-labeled double-stranded oligonucleotide probes in reaction buffer for 20 min at room temperature. Samples were subjected to electrophoresis in 5% nondenaturing polyacrylamide gel and transferred to Biodyne™ BNylon membrane (Cat. No. 77016, Pierce, Rockford, IL). For competition analyses, 100-fold excess of unlabeled probes were included in the binding reaction. For antibody supershift experiments, the reaction mixtures were preincubated with 2 μg of p50 (sc-8414X), p52 (sc-298X), p65 (sc-8008X), c-Rel (sc-272X), RelB (sc-226X) or rabbit IgG (sc-2027) antibody (all from Santa Cruz, CA) for 30 min at room temperature. Biotin-labeled double-stranded oligonucleotides were used as probes listed below: wild-type NF-κB consensus binding sequence: 5′-agttgaggggactttcccaggc-3′ ; wild-type Mcl-1-κB binding sequence: 5′-ggagtcggggtcttccccagtttt-3′, corresponding to the nucleotides of the human Mcl-1 promoter. Unlabeled double-stranded oligonucleotides used for competition analyses were: wild-type NF-κB consensus binding sequence: 5′-agttgaggggactttcccaggc-3′; mutated NF-κB consensus binding sequence: 5′-agttgaggagatctggccaggc-3′ ; mutant Mcl-1-κB binding sequence: 5′-ggagtcg
gtcttccccagtttt-3′; The AP-1 consensus probe was used as a nonspecific competitor for NF-κB: 5′-cgcttgatgagtcagccggaa-3′ . The probes were commercially synthesized by TaKaRa Bio Inc. (Dalian, China). Binding sites were indicated in italics type and mutations were shown in bold type. The mutated nucleotides for NF-κB binding site of human Mcl-1 promoter in EMSA were identical to those of the mutated sequences in the reporter construct.
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) as previously described . Antibodies used for immunoprecipitation were: p50 (sc-8414X), p52 (sc-298X), p65 (sc-8008X), c-Rel (sc-272X), RelB (sc-226X) and rabbit IgG (sc-2027) (all from Santa Cruz, CA). 2 μg of each antibody was used for each immunoprecipitation. The following primers were used in the ChIP assays: human Mcl-1 promoter including the NF-κB binding region, 5′-cacttctcacttccgcttcc-3′ and 5′-ttctccgtagccaaaagtcg-3′ (200 bp).
Statistical analysis was done with the statistical software program SPSS ver.12.0. Results expressed as mean ± S.D. were analyzed using the Student’s t test. Differences were considered significant when P value was <0.05.