1. Cell Culture
Human embryonic kidney cell line HEK293 cell was grown in DMEM medium containing 10% FBS, 100 µg/mL penicillin, and 100 U/mL streptomycin in a 37℃, humidified incubation chamber in the presence of 95% air and 5% CO2. Cells were subcultured in an interval of 2-3 days by detaching cells with 0.05%
trypsin-EDTA and replating in the ratio of 1:2 to 1:4. Murine macrophage cell line RAW264.7 was grown in DMEM medium containing 10% FBS, 100 µg/mL penicillin and 100 U/mL streptomycin in a 37℃, humidified incubation chamber containing 95% air and 5% CO2. Cells were subcultured every 2-3 days.
Cells were from cultural flask detached using gently patting, and subcultivated in the ratio of 1:3 to 1:6.
2. Preparation of Genomic DNA
HEK 293 cells (2×107 cells) were harvested and resuspended in cold PBS (pH 7.4). Cells were washed once with PBS and centrifuged at 3,000×g for 2 min to pellet cells. Following the removal of the supernatant, 400 µL lysis buffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris-HCl (pH 8.0), 0.2% triton X-100, 5 µM DTT, 100mg/mL lysozyme and 20 µg/mL of protease K) were added and resulting mixture was incubated at 50℃ for overnight. Place the digesting mixture on ice after incubation, followed by adding 250 µL saturated NaCl and incubating at 0 ℃ for 10 min. Centrifuge the mixture at 6,000×g for 10 min, and transfer the supernatant to a new 1.5mL eppendorf tube for DNA precipitation. The precipitation of genomic DNA was performed by adding two volumes of 95% ethanol (about 0.4 mL) into the tube, mixed well, and incubated at -20℃ for 2 hr. Decant the upper layer and wash the genomic DNA pellet twice with 1mL cold 75% ethanol. Remove the residue solution and air
dry the DNA pellet by placing tube at room temperature with cup open for 3-5 min. Dissolve genomic DNA in 100µL TE buffer, pH 8.0.
3. Plasmids construction
To generate the human IL-12 p40 promoter/luciferase reporter constructs, we subcloned promoter region (-922 to +35) of IL-12 p40 ( +1 is starting site of transcription ) by PCR by using genomic DNA isolated from HEK293 as template. After purification, the corresponding DNA fragments were ligated into pEasyTTM vector to form pIL12p40/90 Teasy (Appendix 3 and Fig. 3).
The DNA sequence of the subcloned promoter of human IL-12 p40 was confirmed by ABI377 DNA sequencerTM. The serially deleted gene promoter fragments (Fig. 6) of IL-12 p40 were generated by PCR and ligated into pEasy TTM vector. The DNA sequence of the subcloned promoter fragments was confirmed by ABI377 DNA sequencerTM. The promoter fragments were excised from pEasy TTM vectors using SacI/HindIII enzyme and inserted into the same sites of of p3X-NIL-Lucneo [52] after removing 3X-NF-IL6 responding element fragments (Fig. 4 and 5). The resulting reporter vectors for IL-12 p40 promoter activity are illustrated in Fig 3. pIL12p40/5 Leuneo means that the reporter plasmid was inserting from -48 to +33 and containing TATA box only.
pIL12p40/0 Leuneo means that the reporter plasmid was inserting no promoter region. In order to removal the promoter inserting, we excised and religated in NotI/HindIII region from pCDNA3.0 to p3X-NIL-Lucneo.
4. Preparation of reporter plasmids stably transfected cell lines
To generate stably transfected cell lines, the reporter plasmids were electroporated into RAW264.7 cells by using Multiporator (Eppendorf, AG) following manufacture’s instruction. RAW264.7 cells were harvested and pellet by centrifugation at 3000rpm for 5 min under the room temperature.
Wash cells once with DMEM containing 0.5% FCS and calculate the cell density. Resuspend cells by Hypoosmolar Electroporation Buffer (Eppendorf, AG) in a concentration of 5 × 106 cells/mL. Add plasmid DNA to the cells in a final concentration of 25 µg/mL. Transfer cell and plasmid mixture (800 µL) into electroporation cuvettes (4 mm gap width) and carefully avoid the formation of air bubbles. Electroporation was performed under the condition indicated as following : The mode was set at Eukaryotes “µ”；Voltag was at 570 V； “the time constant (µ)” and “No. of pulses” were adjusted to 50 µs and 2, respectively. After the electric shock, the cells were allowed to stand in the cuvette for 5-10 minutes at room temperature. Carefully transfer the cells suspension from the cuvette to T-25 containing 5mL DMEM containing 10%
FCS. The cells were then recovered in a humidified 37 ℃ incubator for about 24 h.
Replacing the conventional cultural medium with selection medium (DMEM containing 10% FBS, 100 µg/mL penicillin and 100 U/mL streptomycin, and 500 µg/mL G418) and cultivated for another 3 days. Some untransfected cells should be eliminated under this constrained selective culturing condition. Harvest the remaining cells and determine the concentration of cells, and replate the cells in the T25 flask. The cells were further grown in the medium containing 750 µg/mL G418 for another 3 days. The selective stress was further constrained by increasing G418 from 750 µg/mL to 1000 ng/mL during the final stage of selection. Replacing selection medium every 2-3 days until the resistant clones cover the surface of cultural well. Transfer the neomycin resistant clones to a new 96-well cultural plate in a cell concentration of 1 cell/well for further amplifying the cell number. The cell number B21, A1 and B30 of neomycin-resistant cell clones were gradually amplified from 12-well plate, a 6-well plate, a T-25 and to a T-75 flask. The stably transfected cell clones were then harvested and transfered to liquid N2
tank for long term storage.
5. Luciferase Assay
Plating various stably transfected cell clones in the 24-well plates in a concentration of 2.5 × 105 cells/well. Following incubation at 37 ℃ for 24 h, cells were treated with cultural medium containing LPS, IFN-γ, drugs, herb extracts, inhibitors or medicines at the indicated concentration. Treatment cells were harvested at the indicated times. Briefly, following removal cultural medium, cells were washed once with 1X PBS, pH7.4, followed by adding 50 µL cell lysis buffer (25 mM Tris-HCl, pH 7.8, 2 mM DTT, 2 mM EDTA, 10%
glycerol, 1% Triton X-100). The resulting cell lysates were stored in the -80
℃ freezer.
The luciferase activity of cell lysate was performed by mixing 50 µL luciferase assay reagent (0.47 mM D-luciferin, 2.67 mM MgSO4, 20 mM Tricine, 0.55 mM ATP, 1.07 mM (MgCO3)4-Mg(OH)2．5H2O, 0.27 mM CoA, 33.3 mM DTT, 0.1 mM EDTA) with 10 µL cell lysate in a 100×12 mm plastics tube. The activity of luciferase was determined on the luminometer (BioOrbit) with a detection protocol: Delay 90 seconds, detect 20 seconds and integrate the relative light units (RLU) detected within 20 seconds. The luciferase activity of each set of samples is normalized using the level constitutively expressed neomycin phospho-transferase (NPT) in each sample.
The protein concentration of cell lysate was determined using Bradford reagent (Bio-Rad) under constant reaction volume of 200 µL containing 4 volumes of didiluted water solution to one volume of Bradford’s reagent.
Following vigously vortexing, the mixture incubated in room temperature for 10
～20 min, determine the absorbance (OD595) at wavelength in 595 nm by Fluostar (BMG) and calculate the protein concentration of cell lysate.
The pIL12p40/90, 56, 40, 25, 20, 10, 7, 5, 0 Lucneo stably transfected RAW264.7 stable clones (RAW(IL12p40/90), RAW(IL12p40/56), RAW(IL12p40/40), RAW(IL12p40/25), RAW(IL12p40/20), RAW(IL12p40/10), RAW(IL12p40/7), RAW(IL12p40/5), RAW(IL12p40/0)) were generated and maintained in the culture medium containing 250 µg/mL G-418 sulfate. The reporter plasmid stably transfected RAW264.7 clones were further tested for their responses to LPS.
(4)Data analysis
After the process of luciferase assay and protein assay, we could obtain the value of the RLU and concentration of cell lysate. Determine the value of RLU divided by the amount of protein in 10 µL lysate. This value (RLU/µg. of prot) indicates the luciferase expressional level and trans-activation activity of the transcription factors in IL-12 p40 promoter region.
Neomycin phosphor-transferase is expression constitutively. In order to normalize copy numbers of different length of promoter reporter plasmids, we check the expression amounts of neomycin phospho-transferase of different stable clones by western blotting (see western blotting). Each sample loads 60 µg of protein amount. The NPT protein and β-actin expression amounts of RAW(IL12p40/90) are used as 100% and others are compared with RAW (IL12p40/90). Thereafter, we use NPT relative percentage to divide β-actin relative percentage to get the NPT/β-actin ratio, which is used as Normalization Index and each RLU/µg. of protein is further divided by Normalization Index.
6. Western Blotting
RAW264.7 cells were grown in a 100 mm dish to about 60-70% confluence before being treated with medium, drugs, stimuli or Chinese herb extracts.
After incubation for the indicated time internals, the cells were harvested and
incubated with cell lysis buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose) at 4 ℃ for 15 min. The total protein of each cell sample was loaded onto a 10% SDS polyacrylamide electrophoresis gel (SDS-PAGE). After electrophoresis at 150 V for 2 hr, the separated protein bands were transferred to a polyvinylidine difluroide (PVDF) membrane through a Mini-Trans Blot Electrophoretic Transfer Cell soak in the presence of Transfer buffer (12.5 nM Tris, 96 mM glycine, 20% methanol, pH8.9). After transblotting, PVDF membrane was blocked with 5% skim milk in PBST (1X PBS with 0.5% Tween 20) at room temperature for 1 h or 4 ℃ for 8～16 h.
The membrane is then incubated with first antibody in PBST with 5% skim milk at room temperature for 1 h or 4 ℃ for 8～16 h. After incubation, the membrane was washed three times with PBST, followed by incubating with horseradish peroxidase conjugated secondary antibody at room temperature for 1 hr. After washing three times with PBST, 1 mL ECL (Amersham) was lay on the membrane for chemiluminescent reaction. The specific protein was visualized by exposing to an x-ray film (Hyperfilm). Protein concentration for SDS-PAGE was determined by Bradford’s assay (Bio-Rad). Stripping buffer we used is containing 65 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM β-mercaptol.
7. Subcellular Fractionation
After treatment, cells were washed twice with cold 1X PBS, pH 7.4, and then suspended in 100 µL buffer I (10 mM Tris-HCl, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and protease inhibitor cocktail) and kept on ice for 15 minutes. After incubation, 25 µL 10% Nonidet P-40 was added followed by a vigorously vortexing for 10 seconds. This procedure will be used to help weakening the cell plasma membrane. The resulting cell lysate was then centrifuged at 14000 rpm for 15 minute at 4 ℃.
The supernatant containing cytoplasmic proteins was removed and stored at -80
℃ freezer. The pellet containing intact nuclear was further subjected to nucleaus preparation. The intact nuclei were resuspended in 50 µL buffer II (20 mM Tris-HCl, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF, 0.1 µg/mL protease inhibitor cocktail) and vigorously vortexing for 15 minutes. Centrifugate the nuclei/buffer I suspension at 14000 rpm for 10 minutes at 4 ℃ to collect the soluble extracted fraction. The nuclear fraction can be stored at least six months at -80 ℃ freezer.
8. Site-directed Mutagenesis
The site-directed mutagenesis toward transcription factor binding sites on
IL-12 p40 promoter were manipulated according to the instruction manual of QuickChangeTM site-directed mutagenesis kit (STRATAGENE). In the beginning, prepare the sample reaction(s) as indicated bellow: 10X reaction buffer, primer pairs for target site (200 ng), PfuTurbo DNA Polymerase (2.5 U), template DNA (75 ng/mL) and 10 mM of dNTP.PCR thermocycle profile was processed at 2 min at 95 ℃, 2 min at 64 ℃ and 10 min at 72 ℃ during 18 cycles. After PCR thermocycles finishing, utilize the restriction enzyme Dpn I to digest the original template, overnight.
Thereafter, transfer the Dpn I-treated DNA for transformation.
9. DNA-sequencing
By Sequencing of all constructed and mutant plasmids was preformed by using Applied Biosystems BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit reagent. The sequencing mixture was prepared as described by manufacture’s instruction. The reaction was performed on a thermal cycles with a cycling condition of 30-sec at 95 ℃ for denaturing, 20-sec at 50 ℃ for annealing, and 4-min at 60 ℃ for elongation. Total 30 cycles were carried out.
The resulting fluorescence-labeled sequence was then product on an ABI 377 automatic sequencer.
10. EMSA (Electrophoretic mobility shift assay)
The probes were prepared by using biotin 3’end DNA labeling kit (PIERCE). Biotinylated probes were added to the binding reaction that also contained 2 µL binding buffer (100 mM Tris, 10 mM DTT, pH7.5), 2% glycerol, 0.5% NP-40, 5 mM MgCl2, 10 mM EDTA, and 2 µL labeled probe ( labeling efficiency is about 70% to 80% ). The binding mixtures were incubated at room temperature for 1.5 h, followed by adding 5 µL loading buffer. For supershift assays, 2 mg of specific Abs were used. For competition analysis, an excess of unlabeled oligocucleotides containing concensus binding sites for transcription factors was added to the binding reaction. Complex formation was proceeding for 1.5 h at 4 ℃. Finally, the samples were fractionated on a 6%
nondenaturing polyacryamide gels in 0.5 X TBE buffer at 100 V for about 2.5 hour. The separated oligonucleotide probe and DNA/protein complex were transblotted to a nylon membrane (S&S) in the same buffer (0.5 X TBE buffer) at 380 mA for 45 min and cross-link the transferred membrane at 120 mJ/cm2 by using UV-light cross linker instrument, SPECTRO LINKERTM (Spectronics Inc.). After blocking at room temperature for 15 min, conjugating with strepavidin-HPR at room temperature for 15~20 min, washing using washing buffer for 6 min 3 times, and substrate equilibrium buffer for 5 min. Then, adding ECL reagent on the membrane for chemiluminescent reaction. The biotin labeled probes were visualized by exposing to an x-ray film (Hyperfilm).
11. MTT assay
Following removal of culture supernatants, cell viability was assessed by replacing medium and adding 500 µg/mL of 3-(4,5-dimethylthiazol-2-yl)
-2,5-diphenyl-tetra-zolium bromide (MTT). After incubation for 3 h at 37 ℃, medium was removed totally and the precipitate formed in metabolically active cells by the reduction of MTT to its insoluble MTT-formazan was dissolved in acidic isopropanol (1N), and the absorbance at 570 nm was determined.