NIH 3T3, RWPE-1, and RWPE-2 cells were acquired from American Type Culture Collection (ATCC, Manassas, VA). NIH 3T3 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA). RWPE-1 and RWPE-2 cells were maintained in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml human recombinant epidermal growth factor (Invitrogen). All culture media contained 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen).
RWPE-1 and RWPE-2 cells were cultured for 24 h and then treated with 0, 20, or 50 μM lunasin for 3 and 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% saponin after lunasin treatment. Cells were stained with an affinity-purified lunasin antibody (1:500 dilution) (5
) followed by an Alexa 488-conjugated goat anti-rabbit antibody (1:250 dilution, Invitrogen). Nuclei were visualized by DAPI staining (Vector Laboratories, Burlingame, CA). Photomicrographs were obtained using a Nikon Eclipse 800 microscope equipped with a digital camera. All images were acquired using the same integration parameters.
Lunasin (89 pmoles, Soy Labs, Fairfield, CA) was incubated with recombinant H4 (rH4) (89 pmoles, New England BioLabs, Ipswich, MA) in 500 μl of histone binding buffer (1X = 20 mM Tris-HCl pH 7.6, 150 mM KCl, 5 mM MgCl2, 10 μg/mL BSA, 0.5% NP-40) at 4°C for 1 h. The peptide binding mixture (lunasin/rH4) was immunoprecipitated by adding a deacetylated H4 (Millipore, Temecula, CA) polyclonal antibody at l:300 dilution or a lunasin polyclonal antibody (Soy Labs) at 1:1000 dilution and incubating at 4°C for 1 h with gentle rocking. For non-specific binding controls, lunasin or rH4 was also incubated with an affinity-purified GST protein (89 pmoles) at 4°C for 1 h and immunoprecipitated with a GST antibody (1:500 dilution, Stressgen, Ann Arbor, MI). A negative control with no antibody added to the peptide binding mixture was also performed. After incubation, 40 μl of Protein-A/G PLUS-agarose slurry (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture and allowed to incubate at 4°C overnight with gentle rocking. Immunoprecipitates were collected by centrifugation at 1000 g at 4°C for 5 min and washed 4 times with 1 ml 1xPBS, pH 7.4. After final wash, the pellet was resuspended in 40 μl of 1x Laemmli sample buffer containing 1 mM β-mercaptoethanol (Bio-Rad, Hercules, CA), boiled for 1 min and centrifuged at 1000 g at room temperature for 5 min. Supernatant was collected and loaded onto 4–20% Tris-HCl gels (Bio-Rad). The pulled-down peptide was detected using western blot analysis with either the lunasin (1:15,000) or the H4 (1:10,000) primary antibody followed by an HRP-conjugated secondary antibody (1:15,000, Pierce, Rockford, IL) and visualized using a SuperSignal West Femto kit (Pierce) and a ChemiDoc XRS+ Imaging System (Bio-Rad).
Coomassie Blue Staining
Recombinant H4 and GST proteins (8 μg) were separated in a 4–20% Tris-HCl gel. Proteins were stained by submerging the gel in a staining buffer containing: 0.2% (wt/vol) Brilliant Blue G250, 20% ethanol (vol/vol), and 0.5% (vol/vol) methanol, at room temperature for 1 h. The gel was rinsed with water and destained with 30% methanol at 4°C overnight.
In Vitro Histone Acetyl Transferase (HAT) Assay
TwoμL lunasin (100 μM) in double distilled sterilized water was mixed with either 10 μg acid-extracted histones from HeLa cells or 1 μg rH4 in a total 25μl volume. Samples were incubated at room temperature for 5 min, on ice for 5 min, and again at room temperature for 5 min. HAT reactions were initiated by adding 10 μl 5x HAT buffer (Millipore), 2 μg acetyl CoA, HAT enzymes (0.5 μg PCAF, 2 μg p300, or 2 μg HAT1A) into the mixtures (50 μl final reaction volume). HAT reaction mixtures were incubated at 30°C with gentle shaking for 10 min (p300 and HAT1A reactions) or 1 h (PCAF reactions). Reactions were stopped by adding 50 μl Laemmli sample buffer (Bio-Rad), boiling for 5 min, and quenching on ice. Twenty-five μl reaction mixture was loaded in each lane of 10–20% Tricine-HCl gels (Bio-Rad), and electroblotted onto nitrocellulose membranes (Bio-Rad). Blots were immunostained with a primary antibody specific to H4 acetylated at K4 (H4K4), K8 (H4K8), K12 (H4K12), or K16 (H4K16) (Millipore) using manufacturer recommended dilutions. An HRP-conjugated goat anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, NJ) was used at 1:10,000 dilution. Chemiluminescence from an ECL Western detection kit (Amersham) was quantified using an Alpha Innotech digital imaging system (Alpha Innotech San Leandro, CA).
RWPE-1 and RWPE-2 cells were grown to 70% confluency in 150 mm plates. Lunasin was added into the medium to a final concentration of 2 μM. For the controls, 1x PBS, pH 7.4, was added to the plates. Cells were incubated for 24 h and lysed with Trizol reagent (Invitrogen) for total RNA purification. cDNA was synthesized from 10 μg of total RNA using a SuperScript Choice system (Invitrogen), then transcribed into cRNA in the presence of biotin-labeled nucleotide triphosphate using T7 RNA polymerase (New England BioLabs). cRNA was purified using an RNeasy mini kit (Qiagen, Valencia, CA) and fragmented at 94°C for 30 min in a buffer containing 0.2 M Tris-acetate, pH 8.1, 0.5 M potassium acetate, and 0.15 M magnesium acetate. Fragmented cRNA was hybridized to HGU133A GeneChip microarrays (Affymetrix, Santa Clara, CA) at 45°C overnight. Hybridization was detected using a confocal laser scanner (Affymetrix). Gene expression levels were normalized and analyzed using robust multichip average (RMA) (13
cDNA was synthesized from 3 μg of total RNA using a SuperScript First-Strand Synthesis (Invitrogen). Two μl of cDNA, diluted 4 fold, was added to the PCR reactions containing TaqMan probes (Applied Biosystems, Foster City, CA). Quantitative RT-PCR (qRT-PCR) was performed in triplicate using an ABI 7900HT (Applied Biosystems) and ACTB expression was used for normalization. Changes in transcript levels were calculated using relative quantification of gene expression following Applied Biosystems’ protocol.
Western blot analysis
RWPE-1 and RWPE-2 were grown to 70% confluency in 100 mm plates. Four hours after seeding, lunasin was added into the medium to a final concentration of 2 μM. For the controls, 1x PBS, pH 7.4 was added to the plates. After 24 h lunasin treatment, cells were lysed with M-PER protein extraction reagent containing 1x Halt Protease Inhibitor cocktail (Pierce). Protein concentrations were determined using Dc protein assay reagents (Bio-Rad). From 25 to 37 μg protein lysate was loaded in each lane of 10–20% Tris-HCl gels with Kaleidoscope markers (Bio-Rad) and electroblotted onto nitrocellulose membranes (Bio-Rad). Blots were immunostained with primary antibodies against THBS1 (1:350) (Abcam, Cambridge, MA), HIF1A (1:500) (Abcam), TOB1 (1:200) (Santa Cruz Biotechnology), PRKAR1A (1:200) (Santa Cruz Biotechnology) and acetylated H4K8 and H4K16 (1:1000) (Millipore) at room temperature for 1 h (THBS1 and HIF1A) or at 4°C overnight (TOB1, PRKAR1A, acetylated H4K8 and H4K16). Blots were also immunostained with a β-ACTIN antibody (1:1000) (Imgenex, San Diego, CA) at room temperature for 1 h for normalization. A HRP-conjugated goat anti-rabbit (Pierce) or a donkey anti-goat secondary antibody (Santa Cruz Biotechnology) at 1:10,000 dilution was incubated with blots at room temperature for 1 h. Chemiluminescence from an ECL western detection kit (Amersham) or a SuperSignal West Femto kit (Pierce) was quantified by using an Alpha Innotech digital imaging system (Alpha Innotech San Leandro, CA).
RWPE-1 and RWPE-2 cells (2x107
cells per assay), treated or mock-treated with 2 μM lunasin for 24 h, were incubated with 1% formaldehyde at room temperature for 8 min to crosslink chromatin and quenched with 1 ml 10x glycine (EZ ChIP, Millipore). After washing with ice cold 1x PBS, pH 7.4, cells were collected by centrifugation at 700 g for 4 min and lysed in 1 ml SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) containing freshly added protease inhibitor cocktail at 1:5 dilution (Millipore). Lysates were sonicated to generate 300–600 bp DNA fragments and divided into two fractions. The first fraction was incubated with an acetylated H4K16 antibody (1μg/reaction; Millipore) at 4°C overnight and the second fraction was incubated with 1x TE buffer as the negative control. Protein A-Sepharose beads were added, incubated at 4°C for 1 h, centrifuged at 3000 g for 1 min before washing for 5 min with ice cold buffers (EZ ChIP, Millipore): 1) low salt wash buffer once, 2) high-salt wash buffer once, 3) LiCl wash buffer once, and 4) TE buffer twice. After washing, beads were treated with RNase and then proteinase K at 55°C overnight. DNA was released from crosslinking by heating the samples at 65°C for 6 h, extracted by phenol/chloroform, precipitated by ethanol, and resuspended in water. DNA from fraction 1 and 2 (10–15 ng) were amplified with a THBS1 CpG island primer set (5′CTTTTCGGATGCTTGCTG3′ and 5′CAGTCTGGGCTCCTCTCTCC3′) and exon 11 primer set (5′CTGAGGCTTCAGTCCCTCTG3′ and 5′AGAGTAAGGAAATCTTGGGGTGTC3′) designed with the Primer 3 software (14
Bisulfite-specific PCR and sequencing
Genomic DNA was isolated from RWPE-1 and RWPE-2 cells with a PureLink Genomic DNA Isolation kit (Invitrogen) and subjected to bisulfite conversion (EZ DNA-Methylation-Direct, Zymo Research, Orange, CA). Bisulfite converted unmethylated cytosines to thymines while methylated cytosines remained intact. Bisulfite-converted genomic DNA was subjected to bisulfite-specific PCR (BSP) using primers specific to the 5′ end of THBS1. The BSP primers, designed using Methyl-Primer Express v1.0 (Life technologies, Carlsbad, CA), were: methylated primer pair (M), 5′TTATTTTTCGTTTTTTCTTCGGTCGTCG3′ and 5′GCGCGCTTTTAAAAAAACGCTCG3′; unmethylated primer pair (UM), 5′TGTTTTTTGTTTGGTTGTTGTTT3′ and 5′CTCACAAACCAACTCAAACACAA3′. PCR was carried out in a 25 μl reaction volume using a Taq PCR Kit (New England BioLabs). PCR products were run on 1.5% ethidium bromide-stained agarose gels, excised, purified using a Gel Extraction Kit (Qiagen), ligated to pCR4-TOPO vector (Invitrogen), and transformed into DH5α E. coli (Zymo Research). Insert-containing plasmids were identified by PCR and EcoRI (New England BioLabs) digestion before sequencing (Davis Sequencing, Davis, CA).
Statistical analysis was performed using paired Student t-test with p <0.05 as the significant standard.