Reagents and antibodies
TSA was purchased from PeproTech. Both polyclonal antibody PAbp68 and monoclonal antibody p68-rgg against human p68 were raised against bacterially expressed His-tagged Cterminal domain of p68 (Invitrogen, Auburn University Hybridoma Facility). Commercial antibodies used in this study were purchased from Santa Cruz (Actin, against human β-actin, HDAC1, GAPDH against human GAPDH, ChIP grade monoclonal against HDAC1, Mi-2, SNAI 1 against Snail1), Cell Signaling Technology (p-Tyr-100, against phosphor-tyrosine, HDAC1, polyclonal against HDAC1, H2A, against human histone 2A), Imgenex (MBD3, ChIP grade), BD Bioscience (E-cadherin and Vimentin), Roche (12CA5, against HA-tag ChIP grade), Abcam (KAT3A/CBP, against CBP) and Upstate (HA, against HA-tag).
Cell culture and RNA interference
SW620 and SW480 cells were purchased from ATCC and grown by following the vendor’s instructions. All DNAs or RNAs transfections were performed using Lipofectamine 2000 by following the manufacturer’s instructions (Invitrogen). For the siRNA experiments, cells were grown to 50% confluence and transfected with siRNA (100 pM). The duplex RNA oligonucleotides for RNAi were purchased from Dharmacon siGENOME™ and SMARTpool®. A duplex RNA oligonucleotides with random sequence (non-targeting, NT) provided by the vendor was included in all siRNA knockdown experiments as negative controls. For transient expression of wild-type p68 or mutants in p68 knockdown cells, the cells were transfected with the indicated plasmid DNA 24 hours after the cells were transfected with siRNA and harvested after an additional 48 hours incubation.
Subcellular extracts, Immunoprecipitation, Immunoblot
All subcellular extracts or whole cell extracts were made freshly after appropriate treatments (indicated in figures). Subcellular extracts were prepared using commercially available cell extracting kit and by following the vendor’s instructions (Active motif). The protein concentration of the extracts was determined using the Bradford assay (Bio-Rad). Immunoprecipitation experiments and immunoblot analyses were performed as described in previous studies (Liu et al., 1998
). The blotting signals were detected using SuperSignal West Dura Extended Duration Substrate (Pierce).
Luciferase reporter assays
Before cells were appropriately treated (indicated in figures), cells were transfected with 1 μg of the indicated reporter plasmid and 0.01 μg of pRL null, which expresses Renilla luciferase from Renilla reniformis as an internal control. The total amount of plasmid DNA was adjusted with pcDNA3-β-Galactosidase. Firefly and Renilla luciferase activities present in cellular lysates were assayed using the Dual-Luciferase Reporter System (Promega). Data were represented as Firefly luciferase activity normalized by Renilla luciferase activity.
Expression of p68s by lentiviral system
Stable overexpression of HA-tagged p68 wild-type or Y593F mutant was carried out using the ViralPower lentiviral expression system (Invitrogen) by following the manufacturer’s instructions. The ORFs of p68 wild type or Y593F mutant with N-terminal HA-tag were cloned into pLenti6/TOPO (Invitrogen). The infections of cells with the lentiviruses that carry pLenti6-p68 were carried out in the presence of 6 μg/mL of polybrene and 10 mM HEPES. Following transduction, the cells were selected by 8 μg/mL of Blasticidin (Invitrogen).
Cells were appropriately treated, then total RNA was extracted using the total RNA extraction kit (Qiagen). The RNA was quantified and then converted to cDNA using the Improm II reverse transcription system (Promega) following the manufacturer’s protocol. The cDNA was then used in the final PCR reaction. The cycles were an initial denaturing of 94 °C for 2 minutes followed by 25 cycles of 94 °C for 15s, 55 °C for 30s, and 72 °C for 1m with an additional extension time of 5m added after the last cycle. Densitometry was performed using the ImageJ program. Primers used were: Snail1 (sense 5′-TCTAGGCCCTGGCTGCTAC-3′ antisense 5′-GCCTGGCACTGGTACTTCTT-3′); B2M (sense 5′- TGCTGTCTCCATGTTTGATGTATCT-3′) antisense 5′-TCTCTGCTCCCCACCTCTAAGT-3′). Primers for B2M were used as PCR and loading controls. Controls for the RT reaction (not shown) contained no template or no reverse transcriptase.
Chromatin Immunoprecipitation (ChIP)
The ChIP experiments were performed using ChIP-IT™ Kit (Activemotif). The precipitation of Snail1 or E-cadherin promoters was determined by PCR using primers spanning nt −680 – −541 of the Snail1 promoter (sense 5′-GGGTGCTCTTGGCTAGCTG-3′ antisense 5′- CTGGAGAGCGTGGCATTG-3′) or nt −164 – +49 of the E-cadherin promoter (sense 5′- GTCACCGCGTCTATGCGAGGCCG-3′, Antisense 5′- GGACACTCGAACGCCTTCAGTCAAGT-3′). TFIIB/Pol II antibodies and mouse IgG were used as control antibodies (included in the kit). ChIP-IT’s negative control primers flank a region of genomic DNA between the GAPDH gene and CNAP1 gene. The primers were provided by the vendor (sense 5′-ATGGTTGCCACTGGGGATCT-3′ antisense 5′- TGCCAAAGCCTAGGGGAAGA-3′).
HDAC activity assay
SW620 cells were lysed in RIPA buffer (Upstate) 48 hr after transfection as described above. The lysate was then diluted in RIPA buffer and HA-tagged proteins immunoprecipitated with anti-HA polyclonal antibody (Upstate). HDAC activities were determined by HDAC Activity Colorimetric Assay Kit (BioVision) according to manufacturer’s instructions. Antibodybound beads were washed in HDAC assay buffer prior to being added to the 96-well plate, to remove immunoprecipitation buffer. Reactions were incubated for 30 min at 37°C with or without the addition of 1 μM TSA. Samples were read in a VICTOR3™ plate reader (PerkinElmer) at 405 nm. Typically each assay was performed 3 times.