Antibodies to MEK1/2 (9122), phospho-MEK1/2 (Ser217/221) (9121), phospho-MLC (Ser19) (3671), phospho-MLC (Thr18/Ser19) (3674), LIMK1 (3842), and phospho-LIMK1 (Thr508)/LIMK2 (Thr505) (3841) were from Cell Signaling Technologies (Beverly, MA). Antibodies against myosin light chain (M4401) and phospho-ERK (M8159) were from Sigma (St. Louis, MO). Antibodies against ERα (sc-543), cdk2 (sc-163), cdk4 (sc-260), cdk6 (sc-177), cyclin A (sc-596), cyclin A1 (sc-15383), cyclin D1 (sc-450), cyclin D3 (sc-6283), cyclin E (sc-481), lamin A/C (sc-6215), LIMK2 (sc-5577), p15 (sc-613), p18 (sc-865), p21 (sc-397G), and p107 (sc-318) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against cdk2 (06-505), cyclin E (07-687), focal adhesion kinase (FAK) (06-543), and Ras (05-516) were from Upstate (Lake Placid, NY). Antibodies against paxillin (610051) and p27 (610241) were from BD Biosciences (San Diego, CA). Additional antibodies used were against ROCK II (ROKα, ab24843; Abcam, Cambridge, United Kingdom), cyclin D2 (RDI-CYCLD2abm-43; Research Diagnostics, Flanders, NJ), and phospho-FAK (Tyr397) (44-624; Biosource, Camarillo, CA). Anti-ERK2 antibody (Ab122) was provided by C. J. Marshall (Institute of Cancer Research, London, United Kingdom). Goat anti-mouse, goat anti-rabbit, and rabbit anti-goat horseradish peroxidase-conjugated antibodies were from Pierce (Rockford, IL).
Generation of KD-ER and ROCK-ER cell lines.
Retroviral constructs for conditionally regulated ROCK II-ER (ROCK-ER) and the kinase-dead version (KD-ER) were constructed as described previously (17
). pBABE puro ROCK-ER and KD-ER plasmids were transfected into BOSC 23 ecotropic retroviral packaging cells with Effectene (QIAGEN) according to the manufacturer's instructions. After 36 h, supernatant was collected and centrifuged at 1,600 rpm for 15 min, and aliquots were stored at −80°C. Exponentially growing NIH 3T3 cells were infected with undiluted retroviral supernatant mixed with 4 μg/ml Polybrene (Sigma) and selected with 2.5 μg/ml puromycin (Sigma) to establish transduced pools.
Cell culture and treatments.
Parental NIH 3T3 cell lines or those expressing either kinase-dead KD-ER and ROCK-ER were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% donor calf serum (DCS; Gibco, Paisley, United Kingdom) at 37°C and 10% CO2. For most experiments, 0.5 × 106 cells were plated onto 100-mm cell culture dishes containing DMEM plus 10% DCS. After 24 h, cells were cultured in serum-free DMEM (serum starved) and treated with or without 4-hydroxytamoxifen (4-HT; 1 μM; Sigma) for 16 h in the presence or absence of pharmacological inhibitors. Inhibitors used were U0126 (Promega) dissolved in dimethyl sulfoxide and used at 10 μM and Y-27632 (Calbiochem) dissolved in water and used at 10 μM. Biocoat fibronectin-coated and poly-d-lysine (PDL)-coated plates were purchased from Becton Dickinson.
For suspension studies, nearly confluent NIH 3T3 ROCK-ER fibroblasts grown in DMEM plus 10% DCS were trypsinized and then placed into suspension in serum-free medium. Cells (1.5 × 106) in serum-free medium were plated onto 100-mm cell culture dishes coated with poly-hydroxyethyl-methacrylate (poly-HEMA; Sigma) and treated with or without 4-HT, in the presence or absence of either Y-27632 (10 μM) or UO126 (10 μM), for 15 h. For studies with actin-disrupting compounds, nearly confluent NIH 3T3 ROCK-ER fibroblasts grown in DMEM plus 10% DCS were trypsinized and 0.5 × 106 cells were plated onto 100-mm cell culture dishes. After 24 h, cells were serum starved and treated with or without 4-HT (1 μM), in the presence or absence of cytochalasin D (CCD; 1 μM; Sigma), latrunculin B (LTB; 0.5 μM; Calbiochem), swinholide A (SWA; 0.05 μM; Sigma), or jasplakinolide (Jasp; 0.5 μM; Molecular Probes), for 16 h.
Cell extraction and immunoblotting.
Following treatment as described above, cells were washed with phosphate-buffered saline (PBS) and then lysed in buffer containing 10 mM Tris (pH 7.5), 5 mM EDTA, 1% (vol/vol) NP-40, 0.5% (wt/vol) sodium deoxycholate, 40 mM sodium pyrophosphate, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 0.025% (vol/vol) sodium dodecyl sulfate (SDS), 150 mM NaCl, and protease inhibitors. Lysates were clarified by centrifugation at 13,000 × g for 15 min. Sixty micrograms of each whole-cell lysate was electrophoresed on SDS-polyacrylamide gel before electrotransfer to nitrocellulose membrane. Blots were probed with antibodies (see above) and appropriate horseradish peroxidase-conjugated secondary antibodies (Pierce), followed by visualization by ECL (Amersham Pharmacia) or SuperSignal West Femto (Pierce) according to the manufacturer's instructions and exposure to Kodak BioMax autoradiographic film. Alternatively, for determining the levels of MLC and phospho-MLC, cells were lysed directly in 3× Laemmli sample buffer. Samples were sonicated and boiled for 5 min, and the supernatant was clarified by centrifugation at 16,000 × g for 5 min. An appropriate volume of each sample was electrophoresed and immunoblotted as described above.
Measurement of Ras activation.
Following treatment as described above, cells were washed with PBS and then lysed by scraping in MLB buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 1% [vol/vol] IGEPAL [CA-630], 1 mM EDTA [pH 8.0], 10 mM MgCl2, 10% [vol/vol] glycerol, 1 mM Na3VO4, 25 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Cleared lysates were incubated for 45 min at 4°C with glutathione-agarose beads coupled to glutathione S-transferase-Raf-1 RBD (Upstate). The beads were washed three times with MLB buffer, and bound proteins were solubilized by boiling with 60 μl of 3× Laemmli buffer and separated by SDS-polyacrylamide gel electrophoresis. Ras-GTP and total Ras were detected by Western blotting with an antibody against Ras.
Cells were fixed for 15 min in 4% (wt/vol) paraformaldehyde (PFA)-PBS and then permeabilized for 15 min in 0.5% Triton X-100-PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with 2% (wt/vol) BSA-PBS for 1 h. Cells were incubated with primary antibodies (1:1,000 dilution) for 60 min, followed by three washes with 2% BSA-PBS and a 60-min incubation with the corresponding fluorochrome-conjugated secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) at a 1:200 dilution. Filamentous actin structures were stained with a 1:250 dilution of Texas Red-conjugated phalloidin (T7471; Molecular Probes, Eugene, OR). For visualization of focal adhesions, cells were fixed and permeabilized in one step with 4% PFA-0.2% Triton X-100-PBS. Primary antibodies were as follows: rabbit anti-MLC (Ser19) (3671; Cell Signaling Technologies), mouse anti-cyclin D1 (sc-450; Santa Cruz), goat anti-lamin B1 (sc-6217; Santa Cruz), and mouse anti-paxillin (610051; BD Biosciences). Coverslips were mounted in Mowiol and visualized with a Bio-Rad MRC1024 confocal microscope.
BrdU analysis by fluorescence-activated cell sorter (FACS).
For cell cycle analysis, serum-starved NIH 3T3 ROCK-ER cells were left untreated or treated with 4-HT (1 μM), either in the presence or in the absence of Y-27632 (10 μM) or U0126 (10 μM), for 16 h. Cells were pulsed with bromodeoxyuridine (BrdU; 10 μM) for 2 h prior to harvesting with trypsin. Cells were fixed with ice-cold 70% ethanol for 20 min at room temperature and then treated with 3 N HCl containing 0.5% Triton X-100 for 20 min. Residual acid was neutralized by incubating the cell suspension with 0.1 M sodium borate (pH 8.5) for 2 min at room temperature. Cells were then incubated with anti-BrdU monoclonal antibody (555627; BD Biosciences Pharmingen) diluted 1:200 for 20 min, followed by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (554001; BD Biosciences Pharmingen) for 20 min. The cell suspension was incubated with propidium iodide (5 μg/ml) for 30 min and then analyzed with a FACScalibur flow cytometer and CellQuest software (Becton Dickinson).
Gene silencing was achieved by transient transfection of small interfering RNA (siRNA) duplexes (Dharmacon, Inc., Lafayette, CO). siRNA duplexes against mouse cyclin A (CCNA2; siGenome duplexes 2 [D-040393-02] and 3 [D-040393-03]), cyclin D1 (CCND1; siGenome duplexes 3 [D-042441-03] and 4 [D-042441-04]), p27 (CDKN1B; siGenome duplexes 3 [D-040178-02] and 4 [D-040178-04]), lamin A/C (D-001050-01; Dharmacon), LIMK 1 (siGenome duplexes 2 [D-043923-02] and 3 [D-043923-03]), LIMK 2 (siGenome duplexes 1 [D-043932-01] and 2 [D-043932-02]), and nontargeting control 1 (D-001210-01) were used. In brief, NIH 3T3 ROCK-ER cells were plated at 1.2 × 105 cells per well of a six-well plate in antibiotic-free medium. Cells were transfected the following day with 4 μl of Lipofectamine 2000 (Invitrogen, Paisley, United Kingdom) and 50 nM siRNA duplexes per well according to the manufacturer's instructions. After 6 h, 20% DCS was added to give a final serum concentration of 10%. siRNA complexes were removed 16 h later, and fresh 10% DCS was added. Twenty-four hours later, cells were trypsinized and plated at 0.5 × 106 cells per 10-cm plate in 10% DCS. The following day, cells were serum starved and then treated with or without 4-HT (1 μM) for 16 h. Cells were harvested as described above.
In vitro cyclin E-CDK2 kinase assays.
Cells were washed twice in ice-cold PBS and lysed in ELB+ buffer (250 mM NaCl, 50 mM HEPES [pH 7.0], 5 mM EDTA, 10 mM β-glycerol phosphate, 10 mM NaF, 1 mM sodium vanadate, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.2% Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Equal amounts of cell lysate (400 μg in 0.5 ml of ELB+ buffer) were incubated with 3 μg of anti-cyclin E (07-687; BD Biosciences) bound to 40 μl of protein A-agarose beads (Upstate) for 90 min at 4°C. Immunocomplexes were washed twice in ELB+, once in 50 mM HEPES (pH 7.4), and once in kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM β-glycerol phosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin). The washed immunoprecipitates were resuspended in 40 μl of kinase buffer containing 2 μg of histone H1 (Upstate), 50 μM ATP, and 5 μCi of [γ-32P]ATP and incubated at 30°C for 15 min. Kinase reactions were stopped by addition of 10 μl of 6× Laemmli buffer. After SDS-polyacrylamide gel electrophoresis and transfer, 32P-labeled histone H1 was visualized by exposure to Biomax film (Kodak). Membranes were subsequently blotted with antibodies against CDK2 (06-505; Upstate) and p21 (sc-397G; Santa Cruz).