Cell culture
The A549 human lung adenocarcinoma epithelial cell line and the Saos2 human osteosarcoma cell line cell were maintained in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin and streptomycin. Cells were routinely cultured at 37°C in a humidified atmosphere of 5% CO2.
RNA interference
Two sets of small interfering RNA (siRNA) duplexes for human Sec6 were prepared as previously described [31]. AllStars Negative Control siRNA (Qiagen, Hilden, Germany) was used as the control. Cells were transfected with RNAi duplexes using Lipofectamine RNAiMAX (Invitrogen). Experiments were performed 48 h after transfection.
Plasmid construction
The full-length human Sec6 cDNA from HSC3 cells was cloned into the HindIII and KpnI sites of the pEGFP-C3 vector (Clontech, Mountain View, CA). GFP-Sec6 was mutated using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to be resistant to siSec6-2 (GFP-Sec6 pm #2) or siSec6-3 (GFP-Sec6 pm #3). The following primers were used: 5’-CACCATCTTGGAGAGGACTGTCACGACCAGAATTGAGGGCAC-3’ and 5’-GTGCCCTCAATTCTGGTCGTGACAGTCCTCTCCAAGATGGTG-3’ for point mutation of GFP-Sec6#2 or 5’-CGTACATGTCCACGCTCACTTCTAACATCATCGCGTGGCTGCGGAAAGCG-3’ and 5’-CGCTTTCCGCAGCCACGCGATGATGTTAGAAGTGAGCGTGGACATGTACG-3’ for point mutation of GFP-Sec6#3. Constructs were confirmed by performing restriction enzyme analysis and sequencing.
Immunoblot analysis
Transfected cells were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1% Triton X-100, and protease inhibitor cocktail). Protein concentration was determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The samples were boiled for 10 min in an SDS sample buffer (New England Biolabs, Inc., Beverly, MA, USA). Equal amounts of protein lysate were separated using SDS-PAGE and electrophoretically transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were then blocked with 5% skim milk in PBS containing Tween 20. The PVDF membranes were immunoblotted using primary antibodies against Sec6 (1:1,000; ab156568; abcam), ERK1/2 (1:1,000; 4695; Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204) (1:1,000; 4370; Cell Signaling Technology), protein kinase d (PKCd) (1:1,000; 9616; Cell Signaling Technology), phospho-PKCd (Thr505) (1:1,000; 9255; Cell Signaling Technology), phospho-PKCd (Thr634) (1:1,000; 9376; Cell Signaling Technology), b-actin (1:1,000; 3700; Cell Signaling Technology), phospho-MEK1/2 (Ser217/221) (1:1,000; 9154; Cell Signaling Technology), MEK1 (1:1,000; 9146; Cell Signaling Technology), MEK2 (1:1,000; 9147; Cell Signaling Technology), PP2A in the A subunit (1:1,000; 2041; Cell Signaling Technology), PP2A in the B subunit (1:1,000; 2290; Cell Signaling Technology), PP2A in the C subunit (1:1,000; 2259; Cell Signaling Technology), dual specificity phosphatase 6 (DUSP6) (1:1,000; 3058; Cell Signaling Technology), ubiquitin (Ub) (1:1,000; 3936; Cell Signaling Technology), cullin3 (Cul3) (1:1,000; 2759; Cell Signaling Technology), ubiquitin protein ligase E3 component N-recognin 5 (UBR5) (1:1,000; 65344; Cell Signaling Technology), target of rapamycin signaling pathway regulator-like 1 (TIPRL1) (1:1,000; ab70795; abcam), GFP (1:1,000; 2956; Cell Signaling Technology), p90RSK (1:1,000; 9355; Cell Signaling Technology), phospho-p90RSK (Thr359) (1:1,000; 8735; Cell Signaling Technology), phospho-p90RSK (Ser380) (1:1,000; 11989; Cell Signaling Technology), phospho-p90RSK (Thr573) (1:1,000; 9346; Cell Signaling Technology), GSK3b (1:1,000; 9315; Cell Signaling Technology), phospho-GSK3b (Ser9) (1:1,000; 9336; Cell Signaling Technology), liver kinase B1 (LKB1) (1:1,000; 3050; Cell Signaling Technology), phospho-LKB1 (Ser428) (1:1,000; 3482; Cell Signaling Technology), vimentin (1:1,000; 5741; Cell Signaling Technology), ZEB1 (1:1,000; 3396; Cell Signaling Technology), Snail (1:1,000; 3879; Cell Signaling Technology), ZO-1 (1:1,000; 13663; Cell Signaling Technology), ZO-2 (1:1,000; 2847; Cell Signaling Technology), ZO-3 (1:1,000; 3704; Cell Signaling Technology). Immunoreactive complexes were visualized using the chemiluminescent HRP Substrate ImmobilonTM Western (Millipore).
Immunofluorescence microscopy
Cells were cultured on a micro-cover glass and rinsed with PBS. They were then fixed with 4% paraformaldehyde, washed with PBS three times, and permeabilized using 0.1% Triton/PBS. The cells were incubated in 10% normal donkey serum for blocking and incubated with the anti-PP2A in the C subunit (1:100; 2038; Cell Signaling Technology) or anti-PP2A in the A subunit (1:100; 2041; Cell Signaling Technology) overnight at room temperature in a humidified chamber. For immunofluorescence analysis, the cells were washed with PBS three times and incubated with secondary antibodies for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain. Images were obtained using a confocal laser-scanning microscope (LSM-700, Carl Zeiss, Jena, Germany).
Immunoprecipitation analysis
Cells were lysed in a lysis buffer. Cell lysates (20,000 mg) were pre-cleaned with protein A sepharose beads (GE health care) for 2 h at 4 °C and incubated with 4 mg PP2A in the C subunit (1:1,000; 2259; Cell Signaling Technology) or alpha4 (1:1,000; 5699; Cell Signaling Technology) antibody overnight at 4 °C and reacted to protein A sepharose beads for 5 h. After being washed four times using the lysis buffer, the immunoprecipitated complex was boiled for 10 min in the SDS sample buffer (New England Biolabs). It was then separated using SDS-PAGE and transferred onto a PVDF membrane (Millipore) and subjected to an immunoblot analysis.
Cell migration assay
A cell migration assay was performed as previously described [33]. After 48 h of transfection with siRNAs, cell migration was assessed using ThincertTM cell culture chambers (Greiner Bio-one, Frickenhausen, Germany). The underside of a polycarbonate membrane insert (8 μm pores) was coated with 20 μg/ml of fibronectin at 4°C overnight. A total of 20,000 cells in serum-free media were added to the insert of each well, and the cells were incubated for 12 h. The cells were fixed with methanol for 10 min and stained with Giemsa (Muto Pure Chemicals, Tokyo, Japan). The number of cells that had migrated to the lower surface of the filters was determined microscopically. The experiments were performed in triplicate and repeated at least three times.
Quantitative real-time reverse transcription-PCR
Quantitative real-time reverse transcription PCR was performed as previously described [32]. The primers used for RT-PCR were: 5′-GCCAACCGCGAGAAGATGA-3′ and 5′-CATCACGATGCCAGTGGTA-3′ for b-actin [34], 5′-TCGTTGTGGTAACCAAGCTG-3′ and 5′-AACATGTGGCTCGCCTCTAC-3′ for the PP2A in the C subunit [35].
PP2A immunoprecipitation phosphatase assay
The PP2A phosphatase assay was performed using a PP2A immunoprecipitation phosphatase assay kit (Millipore) according to the manufacturer's instructions.
Statistical analysis
Data are expressed as means ± standard deviations from three or more independent experiments. Statistical analyzes were performed using Student's t test at a significance level of P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or n.s. (not statistically significant).