Cell line and culture conditions
KB, HCT116, HEK293, and H9C2 cells were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, penicillin and streptomycin. All cells were cultured at 37C in a humidified incubator containing 5% CO2. Mycoplasma testing was performed and found to be a negative.
Phage display
The cDNA corresponding to ZAKα NSLC 414-800 was subcloned into a GST-expressing vector. The GST-ZAKα NSLC 414-800 protein was expressed in E. coli BL21 (DE3) cells and the protein was purified using Glutathione Sepharose 4B resin. The immobilized Sepharose GST-ZAKα NSLC 414-800 protein was subjected to phage display screening by the Ph.D.-C7C phage display peptide library (New England Biolabs) for 1 hour at room temperature. Unbound phages were washed with PBS containing 0.05% Tween-20 (PBST). The bound phages were eluted using 100 mM triethylamine (TEA) and neutralized with 1 M Tris-HCl, pH 7.5. The eluted phages were further amplified in E. coli and amplified phages were again used to bind Sepharose GST-ZAKα NSLC 414-800 protein, undergoing three rounds of biopanning. We wanted to exclude all phages binding to GST. To do this, we incubated the final panning phage solution with 50 µL of 5 µg/ml Glutathione Sepharose 4B GST for 1 hour at room temperature. The unbound phages were incubated with the host bacteria and poured onto a plate and individual plaques were isolated. Phages were isolated and amplified and genome DNA was extracted for sequencing to identify potential ZAKα NSLC 414-800 binding peptides.
Xenografts
Six-week-old nude mice were purchased from National Applied Research Laboratories, Taiwan. Stable expressed PhD10, PhD30, PhD35, or PhD40 KB cells were trypsinized and cell concentrations were adjusted to 1x107 cells/ml. Subsequently, 200 μL of the cell suspension was injected subcutaneously into the back of nude mice through a 26-gauge needle. Each mouse received one injection on both the left and right sides of the back. The mice were killed four weeks after injection by ether inhalation. Tumors were excised, weights were measured, and photographs were taken. Statistical analysis was conducted comparing tumor weights among the different groups. All animal experiments were performed following universally accepted guidelines for animal care and use, and all necessary precautions were taken to minimize pain and distress to the animals. All animal experimental procedures were performed with the approval of the Institutional Review Board of Taichung Veterans General Hospital (IRBTCVGH No: 950727/C06134).
Immunoprecipitation and Western blot analysis
Cell lysates were prepared in IP buffer (40 mM Tris-Hcl pH 7.5, 1% NP40, 150 mM NaCl, 5 mM EGTA, 1 mM DTT, 1mM PMSF, 20 mM NaF, proteinase inhibitors, and 1 mM sodium vanadate). Cell extracts (600 mg) were incubated with 5 ml anti-ZAK (0834) polyclonal antibody (generated in the lab) for 6 h at 4oC. This mixture was then mixed with 20 ml protein-A sepharose suspension and incubated for an additional hour. Immunoprecipitates were collected by centrifugation, washed three times with IP buffer with 0.5% deoxycholate and five times with IP buffer alone, and then subjected to SDS-PAGE.
For the western blot analysis, cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. Total proteins were then separated on SDS-PAGE and the protein bands were visualized using an ECL chemiluminescent detection system (Amersham).
Soft agar assay
2.5 ml of 0.5% Nobel agar was poured into each well of a six-well plate as the bottom agar layer. Cells were suspended in DMEM supplemented with 10% FBS and 0.3% Agar Noble (BD, Difco, MD, USA) and plated onto the bottom layer of Agar Noble. Experiments were performed in six-well plates with 2,500 cells per well in triplicate. Colonies were stained with iodonitro-tetrazolium chloride (INT) solution and counted after two weeks incubation under 37°C and 5% CO2. Colonies were photographed and quantified. The number of colonies was counted under an inverted light microscope at X40 magnification. The data are expressed as mean number of colonies ± standard error (SE) from six fields of three independent wells.
Migration and invasion assay
A Boyden chamber was coated with Matrigel for invasion assays or without Matrigel for migration assays. The chamber was incubated at 37°C for 30 minutes to allow the matrix to solidify. The Boyden chamber assay was performed by filling the bottom well of the chamber with DMEM medium containing 10% FBS. The wells were covered with polyvinylpyrrolidone-free polycarbonate membranes with 8-μm pores (Neuro Probes, Inc.). We then seeded KB stable expressed FLAG vector control, FLAG-PhD30, FLAG-PhD35, or FLAG-ZAKa at 1500 cells/well in serum-free DMEM to the top chamber. The Boyden chamber was incubated for 24 h at 37°C to allow for the migration or for 48 hours at 37°C to allow for the invasion of cells through the membrane into the bottom chamber. After the incubation, the top chamber was removed the top and the top of the membrane was gently wiped with a cotton swab to remove non-migrated/non-invaded cells. Membranes were stained using Giemsa stain. The membrane was rinsed with distilled water to remove excess stain and air-dried. Migrating and invading cells were imaged under a microscope and counted.
In vitro kinase activity assay
Protein kinase assays were carried out using glutathione S-transferase (GST)-RhoGDIb or S20 as a substrate. The GST fusion proteins were bound to ZAKa by incubating them with cellular extracts in the kinase buffer (20 mM Hepes, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl, 2 mM MnCl, 5 mM NaF, 1 mM NaVO, 50 mM NaCl) for 15 min on ice. The beads were pelleted and thoroughly washed with PBST (150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% MeOH, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine) and then incubated with [g-32P]ATP (50 cpm/fmol) in the presence of kinase buffer. After washing the phosphorylated GST-RhoGDIb or GST-S20 with PBST seven times, it was boiled in SDS sample buffer. The proteins were run on a 15% SDS-PAGE. The gel was dried, and phosphorylation of the RhoGDIb or S20 substrate was determined by autoradiography.
Isolation of mitochondria
Cells were cultured to 70-80% confluency. The cells were rinsed twice with ice-cold PBS to remove any remaining media or serum. The cells were then resuspended in hypotonic solution (1.25M sucrose, 10mM MOPS, pH7.2) and allowed to swell for 10 minutes on ice. Swollen cells were suspended in a Dounce and homogenized using six gentle up and down rotating strokes and immediately added to a hypertonic solution (100mM sucrose, 10 mM MOPS, 1 mM EDTA, pH 7.2). The homogenized solution was diluted with two volumes of isolation buffer (75 mM mannitol, 225 mM sucrose, 10mM MOPS, 1 mM EGTA, 0.1% fatty acid free BSA, pH7.2). Sediment nuclei, unbroken cells, and cellular debris were centrifuged at 930 g, 4°C for 5 minutes in a JA-20.1 Beckman rotor. The supernatant was then transferred to Beckman plastic centrifuge tubes. The pellet was added to four ml of isolation buffer, and then homogenization and centrifugation cycles were repeated. All the collected supernatants were combined in Beckman centrifuge tubes and run at 13000 xg under 4°C for 20 minutes. The supernatant was discarded and mitochondrial pellet was resuspend in isolation solution.
Polysome profiling
Cells at 70%–80% confluency were treated with 100 μg/ml cycloheximide for 5 minutes, washed, and scraped off in ice-cold PBS containing 100 μg/ml cycloheximide. They were then collected by centrifugation at 300 xg for 5 min. The isolated mitochondrial fractions were lysed in 425 ml hypotonic buffer (5 mM Tris-HCl pH 7.5, 1.5 mM KCl, 2.5 mM MgCl2), added with 5ml of 10mg/ml cycloheximide and 1 ml of DTT, and vortexed for 5 seconds. The mixture was then added with 25ml of 10% triton X-100 and 25ml of sodium Deoxycholate. Debris was removed by centrifugation at 21,000 xg for 5 min at 4°C. The lysate concentration was measured by UV spectrophotometer. The same amount of lysate was loaded onto 10%–50% (w/v) sucrose gradient and centrifuged at 35000 xg for 2 hours at 4°C in a Beckman ultracentrifuge with the SW40Ti rotor. After ultracentrifugation, the fractions were collected by pipetting of 0.75 ml for each fraction from the top. Absorbance was recorded using a spectrophotometer set at 254nm. All fractions were immediately frozen on dry ice for future study. Proteins were precipitated with 10% trichloroacetic acid (TCA) at 4°C. The samples were spun down at18000 xg for 15 minutes at 4°C and washed twice in ice-cold acetone. Pellets were resuspended in laemmli buffer containing DTT and boiled at 95°C for 5 minutes.
Yeast two hybrid assay
The two-hybrid screen assay was performed as described in the user’s manual (Clontech). Briefly, the full-length ZAK was cloned to the pGBKT7 vector as the bait construct. This vector encoding GAL4 DNA binding domain-ZAK fusion (DNA-BD/ZAK) was transformed into AH109 yeast. This pGBKT7-ZAK yeast was mated with Y187 yeast containing a pretransformed MATCHMAKER human heart cDNA library. The yeast was selected following the manufacturer’s directions. The positive clones were isolated and sequenced.
Isolate mRNA and RT-PCR
Total RNA was isolated from whole cells or purified mitochondrial fractions of KB cells expressing PhD30 or PhD35 using TRIzol (Invitrogen) following the manufacturer's directions. 5 mg of isolated mRNAs was added to the RT-PCR master mix (oligo dT, 10 mM dNTPs, 200 units/μl reverse transcriptase, 5x reaction buffer) at 37°C for 60 minutes. After the reverse transcription reaction, 45 μl of sterile water was added to the mixture. For the PCR reaction, 2 μl of cDNA product from the reverse transcription reaction was used to amplify cDNA with specific primers for CV-ATP5A, CIII-UQCRC2 or actin for 25 cycles. The PCR products were detected by gel electrophoresis.
Pull-down assay
pGex-S20 plasmids were transferred into BL21 (DE3) and positive clones were selected. The positive clone was induced by IPTG. The bacteria were lysed and GST-S20 protein was immobilized by glutathione-sepharose. HEK293 cells were transfected with 10 μg of pEGFP-ZAKa or serial C-terminal deletion mutants following the calcium phosphate method. Cells were lysed in an immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail). 600 μg of the cell lysate was incubated with 20 ml immobilized GST-S20 at 4°C overnight. The pulled-down complex was washed seven times with the immunoprecipitation buffer and the beads were boiled in Laemmli buffer. The complex was run on SDS-PAGE and the GFP conjugated proteins were detected by GFP antibody.