Cell lines and cell culture
Bhas 42 (v-Ha-ras transfected Balb/c 3 T3) cells were purchased from the Health Science Research Resources Bank (Osaka, Japan). Bhas 42 cells are regarded as a model for initiated cells in the cell transformation assay . Bhas 42 cells were cultured in MEM (Thermo Fisher Scientific, Carlsbad, CA, USA) containing 10% FBS (M10F) at 37 °C under 5% CO2 and 95% O2, as described . The v-Ha-ras-transformed NIH 3 T3 (Ras-NIH 3 T3) cells were previously described . Ras-NIH 3 T3 and their parental cells were grown in DMEM containing 10% FBS. These cells were maintained in 75 cm2 vented flask until they reached a density of 70 ~ 80%.
Cells were cultured in 4-well chamber slides. Formalin-fixed cells were permeabilized for 15 min in 0.25% Triton X-100 (in DPBS), blocked, and incubated overnight with the anti-LPAR3 antibody (1:80, Abcam, Cambridge, MA, USA). Images were captured on Axio Imager Z1 fluorescence microscope (Carl Zeiss, Thornwood, NY, USA).
Plasmid DNA, siRNA, and transient transfection
pCMV6-LPAR3 (Myc-DDK-tagged) (CAT# MR226015) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA). pEGFP-LC3 (Addgene #11546), ptfLC3 (Addgene #21074) and pCI-neo-mAtg5 (Addgene #22956) were obtained from Addgene (Cambridge, MA, USA). Murine Lpar3 siRNA was obtained from Integrated DNA Technologies (Coralville, IA, USA). The siRNA sequence was 5′-CUGAAAGGUAGAUCAGUUAAAAACA-3′ (sense). Cells were transiently transfected with the indicated constructs using Lipofectamine 2000.
Measurement of autophagic flux by LC3 conversion
The relevant parameter in LC3 flux assay is the difference in the amount of LC3-II in the presence and absence of lysosomal inhibitors (chloroquine, CQ). Briefly, cells were treated with either vehicle or CQ (30 μM) for 24 h. Western blots were performed with antibody against LC3 as previously described . Images were captured using the Bio-Rad ChemiDoc XRS+ instrument (Hercules, CA, USA). Band intensity values were measured using the Image Lab software, version 5.2.1 (Bio-Rad). LC3-II band intensity was normalized to that of a loading control, β-actin, and ratio further normalized to that in control (untreated). Autophagic flux was calculated by subtracting ratio untreated group from ratio of CQ-treated group.
The tandem RFP-GFP-tagged LC3 fluorescence assay
Cells were transiently transfected with the tandem mRFP–GFP–LC3 reporter plasmid (ptfLC3). After 24 h, the cells were seeded on to 4-well chamber slide before fixation in 10% neutral-buffered formalin. Coverslips were mounted on slides using antifade mounting media. Twenty transfected cells were analyzed for each condition. Yellow puncta are indicators of autophagosomes, whereas red puncta are indicative of autolysosomes in merged image. The number of LC3 puncta was quantitatively assessed withe ImageJ software (NIH, Bethesda, MD, USA).
Fusion of autophagosomes with lysosomes
Autophagosome fusion with lysosome was evaluated by analyzing the colocalization of fluorescent LC3-II with LysoTracker (a fluorescent acidotropic probe for lysosome labeling). For this experiment, cells were cultured in 4-well chamber slides and stained with 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific, Carlsbad, CA, USA). Formalin-fixed cells were permeabilized, and blocked for 1 h in 0.1% Tween 20 and 1% BSA.. The cells were then incubated overnight with anti-LC3 antibody followed by incubation with the FITC-labeled antibody for 60 min. Imaging was performed using the Axio Imager Z1 fluorescence microscope (Carl Zeiss).
Generation of the LPAR3 KO cells with CRISPR/Cas9 system
The plasmid containing single guide RNA sequence designed against exon-2 of the LPAR3 gene was obtained from ToolGen (Seoul, Korea). The sgRNA sequence is as follows: 5′-AAACGTTGACCGTCAACCGCTGG-3′. For the establishment of LPAR3 KO cell lines, pHRS_HumanLPAR3_CMV containing a hygromycin resistance gene (ToolGen) was used. This plasmid expresses a hygromycin resistance protein when the target sequences are cleaved by a nuclease. The knockout clones were generated as previously described . Briefly, NIH 3 T3 cells were cultured in six-well dishes to 70–80% confluence. The cells were cotransfected with 1 μg of the LPAR3 sgRNA plasmid, 1 μg of pRGEN-Cas9-CMV, and 1 μg of pHRS_HumanLPAR3_CMV using Lipofectamine 2000 (Life Technologies). After transfection, these cells were incubated with 150 μg/ml of hygromycin for 2 days. Surviving cells were reseeded at 0.4 cells/well of a 96-well plate for isolation of single-cell clones. The following primer sets were used for confirmation of genome editing by RT-PCR: 5′-ACCGTCAACCGCTGGT-3′ and 5′-CAATTCCATCCCAGCGTGG-3′. GAPDH was used as an internal control.
In vitro two-stage cell-transformation assay
The in vitro two-stage cell transformation assay (CTA) was performed as described . Briefly, cells were plated in 6-well plate at 4000 cells/well. At 24 h after initial culture, culture medium was replaced with medium containing 3-methylcholanthrene (MCA). Four days later, the culture medium was changed to fresh medium containing phorbol 12-myristate 13-acetate (PMA). The culture was continued in the medium containing PMA for 2 weeks, with media changes every 3–4 days. The cells were then cultured in DMEM supplemented with insulin-transferrin-ethanolamine-sodium selenite (ITES, Sigma, St. Louis, MO, USA) plus 2% FBS during the promotion period . Five to six weeks after tumor initiator treatment, cells were stained with a 5% Giemsa solution.
Cell survival assay
Cells were plated in 96-well microtiter plates as 5 × 103 cells/well, and treated with appropriate drugs for 3 or 4 d. Then, the cells in each well were incubated with 10 μL of WST-1 (Takara Bio Inc., Otsu, Japan) for 1 h at 37 °C. The plates were read on a SpectraMax 190 reader (Molecular Devices, San Jose, CA, USA) at 450 nm.
In vitro soft-agar colony-formation assay
To assess growth of transformed cells in soft agar, cells (1 × 104) were seeded in triplicate at 1 × 104 per 60-mm plate in culture medium containing 0.4% agarose over a base layer of culture medium containing 0.7% agarose followed by incubation for 3 weeks. Colonies were counted after staining with crystal violet.
Transwell cell migration assay
Migration assay was performed in triplicate with the 24-well transwell with 8-μm pore filter (Millipore, Billerica, USA). Cells (5 × 104 cells/well) were placed in DMEM medium without serum in the upper chamber, and lower camber was filled with medium containing 10% FBS. The cells were fixed, and stained with 0.05% crystal violet after additional culture for 8 h. The cells adhering to the underside of the filters were analyzed using light microscopy. Bound crystal violet was extracted with acetic acid, and quantified by measuring the absorbance at 550 nm.
Wound scratch assay
Cell monolayers were wounded with a sterile 200 μL pipette tip. Images of the cells were acquired at 0, 4, 8, and 12 h after scratching with an inverted microscope (Zeiss Primo Vert) equipped with the Axiocam 105 camera and ZEN 2.6 software (Carl Zeiss Inc.). The TScratch software was used to quantify open surface areas .
To prepare whole-cell lysates, cells were harvested by scraping into lysis buffer containing 1% Triton X-100 and protease inhibitors. Protein concentrations of cell lysates were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Western blots were performed as previously described . Membranes containing phosphorylated proteins were immunoprobed with the corresponding antibodies: p-MEK, p-ERK, p-Akt, and p-mTOR (Cell Signaling Technology, Danvers, MA, USA). Images were captured using the Bio-Rad ChemiDoc XRS+ instrument (Hercules, CA, USA). Band intensity values were measured using the Image Lab software, version 5.2.1 (Bio-Rad).
Quantitative real-time reverse transcription-PCR (qPCR) analysis
Total RNA extraction, cDNA synthesis, and qPCR were performed as previously described . The primer sets (Bioneer, Daejeon, Korea) designed and used for qPCR analysis are listed in Supplementary Tables S1 and S2. The qPCR data were calculated using the 2−ΔΔCt method  and normalized to GAPDH levels.
Statistical analysis was performed using unpaired t test or by one-way ANOVA followed by Dunnett’s t test. Data are represented as the mean ± standard deviation of at least three independent experiments. The difference was considered significant if p value< 0.05. All analyses were performed using the GraphPad Prism software version 3.06 (GraphPad Software, CA, USA).