Lysophosphatidic acid

Cells were first starved by incubation for 24 hours in serum-free medium. After serum starvation for 24 h, the cells were treated with 44 µM ATX for 24 h, with or without the ATX inhibitor, HA 130 (1 µM), which was added 30 min before ATX treatment. After 24 hrs of incubation, cells were collected in RIPA Buffer (Thermo Fisher Scientific K.K., Kanagawa, Japan) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland), sonicated, and centrifuged. Protein concentrations in the supernatant were determined by a BCA assay using a BCA Protein Assay Kit (Thermo Fisher Scientific K.K., Kanagawa, Japan). Proteins were boiled in Sample Buffer (Thermo Fisher Scientific K.K., Kanagawa, Japan). The same amounts of proteins were loaded onto 4–20% precast polyacrylamide gels (BIO-RAD Laboratories, Hercules, CA USA) and separated by SDS-PAGE. Protein bands were transferred to PVDF membranes (BIO-RAD Laboratories, Hercules, CA USA) and the membranes were immersed in Tris-Buffered Saline with Tween 20 (TBST) containing the first antibody. After washing, the membranes were immersed in TBST containing the second antibody and reacted with ECL substrate (Thermo Fisher Scientific K.K., Kanagawa, Japan). Protein bands were detected by ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL USA). Primary antibodies were: anti-αSMA (Sigma-Aldrich Co., LLC St. Louis, MO USA, 1:1000) and anti-fibronectin (1:1000; Abcam, Cambridge, MA, USA), anti-collagen type I (1:1000; Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated second antibody (1:10,000) was purchased from Thermo Fisher Scientific (Waltham, MA USA). β-tubulin served as the loading control. All membranes were stripped of antibodies using WB Stripping solution and incubated with mouse monoclonal antibody β-tubulin (1:1000), and subsequently with H goat anti-mouse IgG antibody (1:2000). Densitometry of scanned films was performed using ImageJ 1.49 (NIH Bethesda, MD, USA), and results are expressed relative to the loading control (β-tubulin).

The SVF was isolated from iWAT, C3H10T1/2 (CCL-226, American Type Culture Collection) cells, and 293T (CRL-3216, American Type Culture Collection) cells were maintained in DMEM supplemented with 10% FBS. To induce adipocyte differentiation, confluent SVF or C3H10T1/2 cells were cultured with media containing an adipogenic cocktail consisting of 2.5 μM or 1 μM dexamethasone (D4902, Sigma), 5 µg ml⁻¹ insulin (I0516, Sigma), 500 μM isobutylmethylxanthine (I5879, Sigma) and 1 μM rosiglitazone (R2408, Sigma) for 2–3 days. Subsequently, the cells were switched to maintenance medium (culture medium supplemented with 5 µg ml⁻¹ insulin). For Lats1/Lats2 deletion in mature adipocytes differentiated from iAKO SVF, cells were treated with 1 µM 4-hydroxytamoxifen (H7904, Sigma) for 10 days, starting at 7 days after adipocyte differentiation. C3H10T1/2 cells transduced with TAZ4SA-ER^T2 retroviruses containing a puromycin-resistance cassette were subjected to puromycin (2 µg ml⁻¹) selection to establish tamoxifen-inducible TAZ4SA cells. Next, 1 µM 4-hydroxytamoxifen (H6278, Sigma) was treated for 24 h to activate TAZ4SA-ER^T2. For adenoviral expression, cells were infected with adenoviruses encoding TAZ4SA or GFP as described previously^{51 (link)}. For pharmacological inhibition or activation of YAP/TAZ, C3H10T1/2 day-10 adipocytes were treated with 5 μM verteporfin (SML0534, Sigma) for 12 h or C3H10T1/2 day-8 adipocytes were treated with 2 μM lysophosphatidic acid (L7260, Sigma) for 2 h after 12 h of serum starvation. To modulate YAP/TAZ pharmacologically, C3H10T1/2 cells at 8 or 10 days after adipocyte differentiation were treated with either 2 μM lysophosphatidic acid (L7260, Sigma) for 2 h following 12 h of serum starvation or 5 μM verteporfin (SML0534, Sigma) for 12 h, respectively, with ethanol as the vehicle for verteporfin and DMSO for lysophosphatidic acid.

Lipid-binding assays were performed using PIP StripsTM or SphingoStripsTM (lipid–protein interaction assay, Echelon Biosciences, Salt Lake City, UT, USA) as described in [9 (link),22 (link)]. Ppdef1’s lipid binding was detected using polyclonal antibodies generated in rabbits specific to the Ppdef1, DmAMP1 or NaD1 defensins using Gel Doc (Biorad). The PIP Strips are hydrophobic membranes with 100 pmol of the following lipids: Lysophosphatidic acid (LPA), Lysophosphocholine (LPC), Phosphatidylinositol (PI), Phosphatidylinositol (3)-phosphate (PI(3)P), Phosphatidylinositol (4)-phosphate (PI(4)P), Phosphatidylinositol (5)-phosphate (PI(5)P), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine 1-Phosphate (S1P), Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P₂), Phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P₂), Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂), Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃), Phosphatidic acid (PA) and Phosphatidylserine (PS). SpingoStrips are hydrophobic membranes with 100 pmol of the following lipids: Sphingosine (S), Sphingosine 1-phosphate (S1P), Phytosphingosine (PhS), Ceramide (Cer), Sphingomyelin (SM), Sphingosylphosphorylcholine (SPC), Lysophosphatidic Acid (LPA), Myriosine (Myr), Monosialoganglioside-GM1 (GM1), Disialoganglioside-GD3 (GD3), Sulfatide, Psychosine, Cholesterol (C), Lysophosphatidylcholine (LPC) and Phosphatidylcholine (PC). A 5 µL volume of defensin (5 µg/mL) was spotted onto the membrane as a positive control and allowed to dry before blocking.