All cell lines were maintained at 37°C with 5% CO2
. Detailed medium composition is shown in supplementary information
. For serum starvation, cells were incubated in DMEM or DMEM/F12 without other supplements. Detailed culture conditions are described in Extended Experimental Procedures
Cells were transfected with plasmid DNA using PolyJet™ DNA In Vitro Tranfection Reagent (Signagen Laboratories) according to manufacturer's instructions. siRNAs were delivered into cells using RNAiMAX (Invitrogen) according to manufacturer's instructions. Sources of plasmids used are described in Extended Experimental Procedures.
Immunoprecipitation and Immunoblotting
Cells were lysed using mild lysis buffer. Cell lysates were centrifuged for 10 min at 4°C, and supernatants were used for immunoprecipitation. Immunoprecipitates were washed four times with lysis buffer, and proteins were eluted with SDS-PAGE sample buffer. Immunoblotting was performed using standard protocol. Information for antibodies and phos-tag-containing gels is shown in Extended Experimental Procedures.
HEK293A or MCF10A cells were fixed with 4% paraformaldehyde-PBS for 15 min. Following permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C. Secondary antibodies used were Alexa Fluor 488 or 555 (Invitrogen, 1:1000 dilution). Samples were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen) and immunofluorescence was detected using Olympus confocal microscopy. For paraffin-embedded tissues from control or LPAR transgenic mammary glands or tumors, sections were prepared and subjected for immunostaining following deparaffinization, hydration and antigen retrieval. Detailed methods are described in Extended Experimental Procedures.
Immunoprecipitated MST1 was subjected to a kinase assay in the presence of 500 μM cold ATP, 10 μCi [γ-32P]ATP, and 1 μg of GST-Mob. The reaction mixtures were incubated for 30 min at 30°C, terminated with SDS sample buffer, and subjected to SDS-PAGE and autoradiography. Lats1 or HA-Lats2 kinase assays were performed similarly but using GST-YAP as substrates in the absence of [γ-32P]ATP. The phosphorylation of GST-YAP at S127 was determined by immunoblotting using pYAP antibody. Detailed methods are described in Extended Experimental Procedures.
RNA extraction, Reverse Transcription and Real-Time PCR
RNA samples were prepared using RNeasy Plus mini kit (Qiagen). Reverse transcription was performed using iScript reverse transcriptase (Bio-Rad). Real-Time PCR was performed using KAPA SYBR FAST qPCR master mix (Kapa Biosystems). Detailed methods and information for primers are described in Extended Experimental Procedures.
Lentiviral shRNAs were obtained from Sigma Aldrich. ShRNA plasmids together with pMD2.G and psPAX2 were used to produce virus in 293T cells. ON-TARGET plus SMARTpool siRNA were purchased from Dharmacon. Information for shRNAs is described in Extended Experimental Procedures.
Cell proliferation Assay
HEK293A cells (expressing control shRNA or YAP/TAZ shRNA, 2×105) in serum-free media were maintained in the presence or absence of 10 μM LPA for 1, 2 or 3 day. Cell numbers were determined daily using a cell counter (Bio-Rad). LPA was replenished everyday.
Cell migration Assay
Cell migration assay was performed using BD Falcon™ Cell culture inserts for 24-well plates with 8.0 μm pores filter according to manufacturer's instructions. Detailed methods are described in Extended Experimental Procedures.
A detailed protocol for epinephrine injection experiments is shown in Extended Experimental Procedures.
A detailed protocol for lipid extraction from serum is shown in Extended Experimental Procedures.