Cell culture, antibodies and immunoblotting
Caco-2 cells were grown in DMEM containing 10% FBS, 1x non-essential amino acid (Sigma), 1 mM sodium pyruvate (Sigma) and 1x anti-biotic/anti-mycotic (Invitrogen). ARPE19 cells were obtained from ATCC and were grown according to ATCC recommendations.
Antibodies were obtained from the indicated suppliers: rabbit PP6R2, PP6R3 (Bethyl), goat PP2A-A subunit (Santa Cruz), β-actin (Sigma), rabbit E-cadherin (Cell Signaling), mouse E-cadherin, α-catenin, β-catenin (BD Bioscience), species specific secondary antibodies IR 680 (Invitrogen), IR800 (LI-COR), Alexa 488 and Alexa 568 (Molecular Probes). Chicken antibodies for PP6c and PP6R1 and rabbit α-4 antibody were generated as previously described [13, 24]. Mouse anti-PP2Ac monoclonal antibody was provided by Marc Mumby. Cells were lysed in a buffer containing 50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.2% SDS, 0.5% Nonidet P-40, 1 mM Na3VO4, 1 μM Microcystin-LR, and Roche Complete protease inhibitor mixture. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad). The membranes were blocked with 3% skim milk and treated with antibodies as described and quantified by Odyssey infrared scanner and software (Licor Inds.).
Total RNA from Caco-2 cells (0.1 μg) was reverse transcribed in a final incubation volume of 20 μl using a ThermoScript kit (Invitrogen) at 37°C for 1 h. The resulting first-strand cDNA was subjected to quantitative PCR using the SYBR green detection system (Bio-Rad). The sequences of primers of PP6c and GAPDH were adopted from RTPrimerDB primer database (ID: 8029 and 3 respectively). PP6c expression was normalized to an endogenous GAPDH control. The relative quantitative value for PP6c compared with GAPDH was expressed as comparative Ct (2–(ΔCt-Cc)) method.
Luciferase assays of PP6 promoter
The 1500 nt immediately upstream of the PP6 transcription start site on human chromosome 9 was subcloned into pGL3basic. Caco2 cells (4 × 106) were co-transfected in suspension for 3 h using Lipofectamine 2000 (Invitrogen) with 10:1 ratio of reporter plasmids [pGL3basic hPP6-report] (12 ug) and phRL-CMV (Promega) (1.2 ug). Following transfection, cells were plated in triplicate onto 12 well dishes at high density (1 × 106 cells/well) and low density (2 × 105 cells/well). At 72 h following transfection, cells were lysed with the passive lysis buffer (Promega) and luciferase activities were measured with a Berthold LB 953 luminometer using a firefly luciferase assay kit (Biotium). Renilla luciferase activity was measured with 0.09 μM coelenterazine (Biosynth) in 25 mM Tris, pH 7.5, 100 mM NaCl in all samples to normalize for transfection efficiency.
Isolation and analysis of membrane and cytosolic fractions
Caco-2 cells were suspended in MOPS buffer (50 mM MOPS-NaOH (pH 7.4), 125 mM NaCl, 1 mM EGTA, 0.1% 2-mercaptoethanol, and Roche Complete protease inhibitor) and homogenized by glass pestle homogenizer. Homogenates were centrifuged (100,000 g) for 20 min and the supernatant used as the cytosolic fraction. Pellets were resuspended with MOPS buffer and applied on top of 28% sucrose layered on 50% sucrose. After 40 min centrifugation (100,000 g), the 28/50% interface was collected and washed with MOPS buffer by centrifugation. The resulting pellets were solubilized in MOPS buffer containing 1% Nonidet P-40 and after 15 min centrifugation (16,000 g), the supernatant was used as the membrane fraction. Antibodies were bound to protein G-agarose beads and covalently cross-linked by dimethyl pimelimidate treatment (Harlow and Lane ). Cytosolic and membrane fractions of Caco-2 cells were incubated with antibody-coupled beads. Normal IgG coupled to beads was used as control.
In vitro PP6 binding assay
Binding assay was performed by incubating S-tag PP6c on S protein agarose beads with in vitro translated 35S labeled Flag E-cadherin C-terminal segment. Human PP6c mRNA sequence was inserted into pTriEX4 vector. pTriEX4 PP6c or pTriEX4 empty vectors were transfected into HEK293T cells and cultured for 2 days. Cells were lysed with MOPS buffer (50 mM MOPS-NaOH (pH 7.4), 125 mM NaCl, 1 mM EGTA, 0.1% 2-mercaptoethanol, and Roche Complete protease inhibitor) containing 1% Nonidet P-40. S-tag PP6c was pulled down by S-protein agarose (Novagen) at 4°C for 4 h. Precipitates were washed to remove associated proteins with 2 M Urea/MOPS buffer twice, with 1 mM ATP/1mM MgCl2/MOPS buffer once, and then with MOPS buffer once. pGEX5X-1 Rat E-cadherin cytosolic fragment was provided by Dr. Barry M. Gumbiner and subcloned into pcDNA3 Flag vector. For in vitro transcription and translation, TNT T7 coupled Reticulocyte Lysate System (Promega) was used with 35S methionine. Empty pcDNA3 Flag vector was used as a control. After 2 h at 4°C beads were washed with MOPS buffer and binding proteins were separated by SDS-PAGE and S35 bands visualized with a Phosphor-Imager (Molecular Dynamics).
Virus production and PP6c knockdown
Short hairpin RNA (shRNA) targeting PP6c (5' CCAGAACGACAACGCCATATT 3’) was inserted into TRIPZ vector (Open Biosystems). Lentivirus for expressing of PP6c shRNA and non-coding shRNA were packaged by co-transfecting plasmids of lenti-vector, psPAX2 and VSV-G into HEK293 cells (Addgene). The virus-containing medium was collected at 48–72 hrs. Adenovirus was prepared using the system developed by He et al. . Briefly, the fragment containing the shRNA, inducible tetracycline responsive RNA Polymerase II promoter and turboRFP was excised and inserted in the shuttle vector, pAdTrack. The resulting plasmids were cotransfected with the adenoviral backbone plasmid, pAdEasy-1, into BJ5183 bacteria. Recombined plasmid was selected and transfected into HEK 293 cells to generate virus, which was amplified and then purified by CsCl gradient ultracentrifugation to create a high titer viral stock. Viruses encoding the inducible expression region and turboRFP from TRIPZ vector were used as a control.
Caco-2 cells were cultured to 80% confluence and infected with control shRNA or PP6C shRNA virus. Approximately 6–8 hours post-transduction, media was replaced containing doxycycline at a concentration of 1 μg/ml. After inducing expression for 4 days, the cells were treated without or with IC261 (10 μM) for 4 h and then fixed for fluorescent microscopy.
Co-expression of myc-E-cadherin and shRNA
The AgeI restriction site in the TRIPZ vector containing the non-coding shRNA and PP6c shRNA was used to ligate blunt-ended fragments encoding mouse E-cadherin wild type (WT) and S846A tagged with myc epitope. Lentiviruses were produced by transfecting TRIPZ vector, psPAX2 and pCMV-VSVG in HEK293-LT cells and virus-containing media were collected at day 2 and 3. Caco-2 cells were infected with lentivirus and selected with 5 μg/ml puromycin. Cells with stable expression were induced by doxycycline (1 μg/ml) when they were confluent. After inducing expression for 4 days cells were fixed by methanol/acetone (1:1) for immunofluorescence staining.
Caco-2 cells were fixed by methanol/acetone (1:1) and permeabilized with 0.5% Triton X-100 for 20 min. The specimens were blocked with 10% goat serum for 1 hr at room temperature. The primary antibodies (E-cadherin from BD Biosciences 1:200, occludin from BD Biosciences 1:400, ZO-1 from Upstate 1:200, β-catenin from BD Biosciences 1:200 and c-myc from Santa Cruz 1:200) were incubated at 4°C for overnight. Secondary anti-rabbit or anti-mouse antibodies were conjugated with Alexa fluor-488 or 647 (Invitrogen). The immunofluorescent images were captured by Leica SP5 X White Light confocal microscope. Fluorescence intensity was measured by line scanning with software in the Leica application suite. Results of line scan were plotted with Gaussian fit and Gaussian full width at half maximum (FWHM) was calculated.
The results are expressed as the means ± S.E. Comparisons between groups were performed by one-way ANOVA, followed by Student-Newman-Keuls test. For all of the analyses, a probability value of p<0.05 was considered as statistically significant.