Anti-HDAC2 antibody was procured from Active Motif (Carlsbad, CA, USA) and Anti-NF-κBp65, anti-phospho NF-κBp65, anti-acetyl CBP/p300 and anti-phospho IκBα were procured from Cell Signaling Technology (Beverly, MA, USA), respectively. Anti-acetyl p65 was procured from Abcam Inc. (Cambridge, MA, USA). Transcription ELISA kit (TransAM NF-κBp65), HAT and HDAC assay kit (colorimetric) were purchased from Active motif (Carlsbad, CA, USA) and Biovision (Mountain View, CA, USA), respectively. Anti-p300 antibody and Milliplex ™ MAP ELISA Kit were purchased from Millipore (USA). Curcumin was purchased from Sigma (St. Louis, MO,USA). The BCA™ protein assay kit was purchased from Pierce. Novex pre-cast Tris-Glycine gels were obtained from Invitrogen (Carlsbad, CA,USA). All other chemicals, unless otherwise stated, were obtained from Sigma (St. Louis, MO, USA).
Cell culture and treatment with curcumin
Human monocytic THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA). THP-1 cells were cultured in RPMI medium containing 10% fetal bovine serum and 1% antibiotics at 37 °C and 5% CO2. Curcumin (dissolved in DMSO) was used for the treatment of cells. The final concentration of DMSO used was 0.1% (v/v) for each treatment. THP-1 cells (1 × 105 cells/ml) were cultured in presence of osmolar control (19.5 mmol/l mannitol) or normo glycemic (NG, 5.5 mmol/L glucose) or hyperglycemic (HG, 25 mmol/L glucose) conditions in absence or presence of curcumin (1.5-12μM) for 72 h after which media was saved for cytokine release measurement and cells were washed with phosphate-buffered saline (PBS) and then harvested.
Cytokine release measurement
Cytokines were measured with a Milliplex ™ MAP Assay Kit (Millipore,USA) according to the manufacturer's instructions. Values were calculated based on a standard curve constructed for the assay.
Trypan blue exclusion assay for cellular viability
The cytotoxicity of curcumin to cultured high glucose-induced THP-1 cells was measured by trypan blue assay. The effect of curcumin on growth inhibition was assessed as the percentage of inhibition in cell growth where vehicle-treated cells were taken as 100% viable. Cytotoxic effect by curcumin (0.5 to 12.5μM; 72h) treatment was not observed. Curcumin treatment was cytotoxic at ≥ 25μM. To be in the physiologic range (0.75-10μM), only concentrations of curcumin <6μM were used for all further experiments.
Detection of transcription factor NF-κBp65 using ELISA
Following treatment of cells with various concentration of curcumin for 72 h, cells were harvested and nuclear lysates were prepared. The commercially available kit for NF-κB/p65 (Active Motif, Carlsbad, CA) contains the specific oligos with the specific consensus sequence for NF-κB/p65 binding. Five micrograms of nuclear lysate protein from each group was taken for quantification of NF-κB activity. The experiment was done according to the manufacturer's instructions. Absorbance was taken at 450 nm by using ELISA reader (Multiscan MCC/340, Fisher Scientific).
Measurement of HDAC and HAT activity using ELISA
Following treatment to cells with various concentrations of curcumin for 72 h, cells were harvested and nuclear lysates were prepared. 10 μg and 50 μg of nuclear lysate protein from each group were taken for determination of HDACs and HATs activity, respectively. The experiment was done according to the manufacturer's instructions. Absorbance was taken at 405 nm and 440 nm.
Preparation of Nuclear and Cytoplasmic Lysates
After treatment of cells with curcumin the medium was aspirated and the cells washed twice in PBS (10 mM, pH 7.4). Nuclear lysates were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). The lysates were collected and cleared by centrifugation, and the supernatant was aliquoted and stored at −80°C. The protein content in the lysates was measured by BCA protein assay (Pierce), as per the manufacturer's protocol.
Western Blot Analysis
For Western blot analysis, 12 μg of protein from each treatment was resolved over 10% Trisglycine polyacrylamide gels (Novex), transferred onto nitrocellulose membranes, and subsequently incubated in blocking buffer [5% nonfat dry milk/1% Tween 20; in 20 mmol/L TBS (pH 7.6)] for 2 h. The blots were incubated with appropriate primary antibody (HDAC 2, p300, acetylated p300/CBP, p-NF-κBp65, NF-κBp65 and acetylated p65) washed, and incubated with appropriate secondary horseradish peroxidase–conjugated antibody (Amersham Biosciences). The blots were detected with chemiluminescence (ECL kit, Amersham Biosciences) and autoradiography, using XAR-5 film (Eastman Kodak). Equal loading of protein was confirmed by stripping the blots and reprobing with TATA binding protein (Abcam Inc, MA, USA)
Total RNAs were extracted from cultured cells using TRIZOL reagent (Invitrogen). Template cDNAs were obtained by reverse transcription (RT) using QuantiTect reverse trasnscription kit (Qiagen). Detection of cDNAs was done by PCR reactions using primers designed for candidate transcript detection. HDAC2: sense:5′-GGGAATACTTTCCTGGCACA-3, Antisense;5′ACGGATTGTGTAGCCACCTC-3′,p300:forward:5′-GGTCCACTCCAATCCAG-3′, Reverse: 5′CTCAAGATGTCTCGGAA-3′. Real-time RT-PCR experiments were done in triplicate using SYBR Green chemistry and a Mastercycler ep gradient S (Eppendorf). The quantity in each sample was normalized to the level of GAPDH transcripts. RT-PCR data were calculated by Delta-Delta CT method.
After treatment of cells with curcumin, the cells were centrifuged and medium was aspirated. Cells were washed twice in PBS (10 mM, pH 7.4) and placed on L-lysine coasted slides, the slides were air dried, fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4°C with HDAC2, p300, acetylated p65 antibody (1:1000 dilution). After being air dried, slides were incubated with appropriate secondary antibody for 60 min. The slides were washed as described above, air dried, mounted with mounting medium, and then examined with a fluorescence microscope at 400 × magnification. In order to measure immunostaining intensity of HDAC2, p300 and acetylated p65, images were captured with a Nikon eclipse TE200 camera (Japan). The signal intensity was measured using ImageJ software.
Chromatin immunoprecipitation assays
ChIP assays were performed using Chip-IT™ Express (Active Motif) according to the manufacturer's instructions. After treatment of cells with curcumin, the cells were centrifuged and medium was aspirated. Cells were washed twice in PBS (10 mM, pH 7.4) and fixed with fresh Fixation solution (37% formaldehyde) for 5 sec at room temperature, followed by glycine stop-fix solution. Cells were washed twice with cold PBS, PBS was poured off and discarded and cells were scraped, pelleted by centrifugation for 10 min at 2,500 rpm at 4°C. Cells were resuspended in 1 mL of ice-cold lysis buffer followed by 30 min incubation on ice. Pellets were spun down for 10 min at 5,000 rpm. Chromatin were sheared using 25% power sonication using our optimized condition (10 pulses of 20 sec each with a 30 sec rest on ice between pulses) to an average DNA size of 600 bp and lysates were cleared by centrifugation at 14,000 for 10 min at 4°C. For each ChIP, one-tenth of the total sonicated chromatin volume (500 μL) was used. Immunoprecipitations were performed overnight at 4°C with 5 μL of the p300 antibody. Chromatin–antibody complexes were captured to magnetic beads (25 μL) and chromatin was eluted as described in manufacturer's instructions. The cross-links were reversed and DNA purified by proteinase K. DNA was analyzed by PCR.
DNA concentration was measured spectrophotometrically at 260 nm. DNA was subjected to PCR. The antibodies against p300 were purchased from Upstate Biotechnology. Primer sequences for the amplification of IL-6 and TNF-α. IL-6 were: Forward: 5′-TTGCGATGCTAAAGGACG-3′ and reverse; 5′- TGTGGAGAAGGAGTTCATAGC- 3′. TNF-α forward:5′-CCCTCCCAGTTCT AGTTCTATC-3′ and reverse:5′-GGGGAAAGAATCATTCAACCAG-3′. PCR was performed after a 4 min denaturation at 94°C, and repeating the cycles of; 94°C, 55°C and 72°C each for 40 sec; the number of cycles was specific for each primer set. PCR products were electrophoresed in a 1.5% agarose gel containing ethidium bromide.
Each experiment was performed at least three times. Results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test and statistical significance is expressed as *, P < 0.05, **, P < 0.01.