HEK293T, A498, and MCF-7 cells were maintained in DME supplemented with 10% FBS. MCF-10A cells were cultured in DME/F12 supplemented with 5% horse serum, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 20 ng/ml EGF, and 10 µg/ml insulin (Debnath et al., 2003
). HK2 cells were cultured in keratinocyte serum-free medium supplemented with 0.05 mg/ml pituitary extract and 5 ng/ml EGF.
HEK293T cells were transiently transfected with the indicated constructs using the calcium phosphate method. Cells were disrupted in a hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP-40, and protease inhibitors). After centrifugation at 3,000 g for 10 min, the cytoplasmic fraction was taken from the supernatant. The pellet was washed with PBS and, after centrifugation for 10 min at 10,000 g, treated with a hypertonic buffer (5 mM Hepes, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, 300 mM NaCl, and protease inhibitors) to yield the nuclear fraction. Both fractions were analyzed by Western blotting.
MCF-10A cells were seeded onto coverslips and transfected with the indicated plasmids using transfection reagent (Genejuice; EMD). 24 h afterward, cells were rinsed with PBS several times and fixed with 4% PFA for 10 min. After blocking with 5% normal donkey serum and 0.1% Triton X-100 in Dulbecco’s PBS, cells were sequentially stained with the indicated antibodies (primary antibodies: rabbit anti-FLAG [pAb] and mouse anti-V5 [mAb]; secondary antibodies: Cy3-conjugated anti–rabbit IgG and Cy2-conjugated anti–mouse IgG obtained from Jackson ImmunoResearch Laboratories, Inc.). Afterward, the coverslips were mounted with Prolong gold with DAPI (Invitrogen) and subjected to immunofluorescence microscopy. Pictures were taken with a microscope (objectives: Plan Apochromat 20×/0.8 NA differential interference contrast, EC Plan Neofluar 40×/1.3 NA oil differential interference contrast, and C-Apochromat 63×/1.22 W; Axiovert 200; Carl Zeiss) equipped with a camera (AxioCam MRm; Carl Zeiss) and an imaging system (ApoTome; Carl Zeiss) using Axiovision 4.8 software for acquisition and subsequent image processing (Carl Zeiss). Figures were assembled using Macromedia Freehand 11 software (Adobe). About 200 cells were counted and quantified in regard to the localization of TAZ and YAP with or without NPHP4 coexpressed in the same cell. P-values were calculated using Fisher’s exact test.
The luciferase reporter plasmid (pGBD-Hyg-Luc) was transfected together with an activator plasmid (pGal4-TEAD), pGL4.74 (Promega) for normalization, and the indicated expression plasmids (TAZ, YAP, NPHP4, Lats1, and the control empty pcDNA6) into HEK293T cells in a 96-well format using Lipofectamine LTX (Invitrogen) as a transfection reagent. The total amount of DNA was always adjusted with empty pcDNA6. Renilla luciferase and firefly luciferase activities were measured by using a reporter assay system (Dual Luciferase; Promega) in a luminometer (Mithras LB 940; Berthold) 24 h after transfection. For the Lats1 siRNA experiment, the indicated plasmids and siRNAs were cotransfected into HEK293T cells. The measurement was performed 48 h after transfection. Transfections and measurements were performed in triplicates for each single experiment, and each experiment was repeated at least three times. Error bars shown in the figures represent SEM. P-values were calculated using an unpaired Student’s t test. Equal expression of the transfected proteins was confirmed by Western blot analysis.
HEK293T cells were transiently transfected using the calcium phosphate method, and the total amount of DNA was always adjusted with empty pcDNA6. The following day, cells were harvested with ice-cold PBS. A small aliquot of this cell suspension was taken, and the cells of which were lysed directly in SDS-PAGE sample buffer (whole cell lysate). The harvested cells were lysed in a 1% Triton X-100 buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, 2 mM Na3VO4, and complete protease inhibitors [PIM; Roche]) for 15 min on ice. After centrifugation at 15,000 g for 15 min at 4°C and ultracentrifugation at 100,000 g for 30 min at 4°C, the supernatant was incubated at 4°C for 2 h with the anti-FLAG (M2) antibody covalently coupled to agarose beads (Sigma-Aldrich) or with 1 µg of the appropriate first antibody and 20 µl protein G–Sepharose beads (GE Healthcare). Before the addition of antibodies, a small aliquot of each supernatant was preserved and diluted with 2× SDS-PAGE sample buffer for later Western blot analysis (lysate). The beads were washed extensively with lysis buffer, and bound proteins were resolved by SDS-PAGE, blotted on to polyvinylidene fluoride membranes, and visualized with enhanced chemiluminescence after incubation of the blots with the respective antibodies.
Plasmids, reagents, and antibodies
TAZ, YAP, Salvador, and Lats1 cDNAs were provided by M. Yaffe (Massachusetts Institute of Technology, Boston, MA) and M. Sudol (Geisinger Clinic, Danville, PA). The TAZ 4SA mutant was generated using mutagenesis (QuickChange; Agilent Technologies) to mutate the serine residues in positions 66, 89, 117, and 306 to alanine. The GAL4-TEAD reporter system (pGBD-Hyg-Luc and pGal4-TEAD) was purchased from Biomyx. NPHP4 was cloned from a human kidney cDNA library. NPHP1 was cloned from a human kidney cDNA library into a modified pcDNA6 vector using standard PCR cloning techniques and has been described previously (Otto et al., 2003
; Schermer et al., 2005
). siRNAs used in knockdown experiments were directed against the following sequences: NPHP4 #5, 5′-AAGCAACGAGATGGTGCTACA-3′; NPHP4 #6, 5′-CAGATCTCGGGTCATCTCAAA-3′; TAZ, 5′-AGGTACTTCCTCAATCACA-3′ (previously described and validated by immunoblotting with endogenous TAZ in MCF-7 cells; Chan et al., 2008
); scrambled/control, 5′-AAATGTACTGCGCGTGGAGAC-3′; Lats1 #1, 5′-GGAGTGTTACTCCTCCACC-3′; Lats1 #2, 5′-GGTTCTGAGAGTAAAATTA-3′, Lats1 #12, 5′-CAGCAGCGTCTACATCGTAAA-3′; and Lats1 #13, 5′-TTGGTGAGTGTTCCTAGGCTA-3′. siRNA strands were purchased from Biomers or QIAGEN (Lats1 #12 and Lats1 #13 siRNA). siRNAs against NPHP4 and TAZ have been tested by qPCR (Fig. S3 a), and the siRNA against TAZ has been previously described (Chan et al., 2008
Antibodies were purchased from Sigma-Aldrich (anti-FLAG/M2, anti–β actin, anti-WWTR1/TAZ, and antitubulin), Cell Signaling Technology (anti-Lats1, antiphospho-Lats1 Ser-909, antiphospho-YAP Ser-127, and anti-MST1), AbD Serotec (anti-V5), Millipore (anti-V5), Abcam (antifibrillarin), and Santa Cruz Biotechnology, Inc. (anti-GFP and anti–14-3-3).
MCF7 cells were transfected with the indicated siRNAs (final concentration of 20 nM) using a transfection reagent (Oligofectamine; Invitrogen). 72 h after transfection, cells were harvested in lysis reagent (QIAzol; QIAGEN), and RNA was isolated using the phenol-chloroform method. After DNase treatment (Invitrogen), the reverse transcription reaction was performed with a High-Capacity cDNA kit (Applied Biosystems). TaqMan assays (Applied Biosystems) were used to evaluate CTGF (Hs00170014_m1) levels. ActB (4326315E), HPRT1 (Hs02800695), and 18SrRNA (4319413E) served as endogenous controls. The efficiency of the NPHP4 siRNAs was confirmed using a TaqMan assay (Hs00296416_m1; Applied Biosystems), the TAZ siRNA was validated using SYBR green qPCR (TAZ forward primer, 5′-ACCCACCCACGATGACCCCA-3′, and TAZ reverse primer, 5′-GCACCCTAACCCCAGGCCAC-3′), and HPRT1 served as an endogenous control (HPRT1 forward primer, 5′-TGACACTGGCAAAACAATGCA-3′, and HPRT1 reverse primer, 5′-GGTCCTTTTCACCAGCAAGCT-3′). All qPCR experiments were performed on a real-time PCR system (7900HT; Applied Biosystems) and repeated at least three times. Error bars shown in the figures represent SEM. P-values were calculated using an unpaired Student’s t test.
MCF-7, A498, and HK2 cells were transfected with the indicated siRNAs (final concentration of 20 nM) using Oligofectamine and processed 72 h after transfection. The MCF-7 cells were serum starved for 5 h followed by 30-min BrdU labeling in complete growth medium. After fixation in 100% methanol for 20 min, cells were stained with the BrdU Labeling and Detection Kit I (Roche). Nuclei were counterstained with DAPI. Pictures were randomly taken with a microscope (Axiovert 200) and the Axiovision software. At least 400 cells were counted for each point within each single experiment.
The A498 and HK2 cells were labeled with BrdU in complete growth medium for 3 or 12 h, respectively. Afterward, the cells were fixed with 70% ethanol overnight after treatment with 2 N HCl/0.5% Triton X-100 and 0.1 M sodium tetraborate. Cells were stained with a mouse anti-BrdU antibody (Sigma-Aldrich) and Dylight 488–conjugated anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). DNA was counterstained with propidium iodide (Sigma-Aldrich), and cells were analyzed by FACS. The amount of BrdU + cells for each condition was calculated in comparison to the scrambled siRNA–treated control cells. Error bars shown in the figure represent SEM. P-values were calculated using an unpaired Student’s t test.
HEK293T cells were transfected with the plasmids indicated in the figures using the calcium phosphate method. 2 × 107 cells were cross-linked with 1% formaldehyde (Merck) for 10 min at RT. Cross-linking was quenched by cold PBS. Cells were scraped into 50 ml PBS containing protease inhibitors (PMSF and apotinin) and pelleted for 5 min at 300 g at 4°C. Cells were lysed, and the lysate was sonicated to a DNA fragment with a length of ~1 kbp. Lysates were cleared by CL4B200 beads (Sigma-Aldrich). Then, 2 µg anti-FLAG antibody and anti–β actin as a negative control was added for overnight immunoprecipitation. 60 µl of preblocked GammaBind G Sepharose beads (GE Healthcare) was added for 1 h at 4°C with rotation. After washing, beads were eluted in 500 µl of buffer. The eluate was incubated for 65°C overnight to reverse cross-linking followed by proteinase K (Roche) digestion and DNA recovery with a PCR purification kit (QIAquick; QIAGEN). DNA was eluted in 50 µl H2O, and 4 µl of the eluate was used per well as the template for real-time PCR using SYBR green–based master mix (Applied Biosystems). Primers for the human CTGF promoter region close to the TAZ response element had the following sequences: 5′-GAGACTGCATCCTGAGTCACAC-3′ (forward primer) and 5′-GGCTCTTGAAACTCTCCAAAGA-3′ (reverse primer).
Online supplemental material
Fig. S1 shows that NPHP1 and NPHP4 do not interact with MST1 and demonstrates the dose-dependent effect of NPHP4 on TAZ signaling. Fig. S2 shows that NPHP4 does not affect autophosphorylation of Lats1 and that the effect of NPHP4 on TAZ activity depends on the presence of Lats1. Fig. S3 shows the validation of the siRNA against NPHP4 and TAZ by qPCR and the reduced cell proliferation after knockdown of TAZ or NPHP4 in renal cell carcinoma cells (A498) and in human kidney epithelial cells (HK2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201009069/DC1