S1pr2

Tissues samples were harvested and lysed in ice-cold lysis buffer (1% Nonidet P-40, 0.5% sodium cholate, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 20 mM HEPES, 3 mM MgCl, 1 mM PMSF, 20 mM b-glycerophosphate, 1 mM NaF and 1 mM sodium orthovanadate; pH 7.4) and sonicated (20 kHz; 0°C; 30 sec). Following centrifugation at 13,225 × g for 20 min at 4°C, protein concentrations were assayed using Bradford protein assay reagent (Bio-Rad Laboratories, Inc.) with BSA as a standard. Total protein (30 µg) was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore). The membranes were blocked with 5% non-fat milk in TBST [10 mM Tris (pH 7.4), 100 mM NaCl and 0.5% Tween 20] for 1 h at room temperature and incubated with the primary antibody S1PR2 (Santa Cruz Biotechnology, Inc.; cat. no. sc-365589; 1:1,000). Protein bands were normalized to GAPDH and detected with anti-GAPDH (Santa Cruz Biotechnology, Inc.; cat. no. sc-47724; 1:1,000). Immunoreactive bands were detected using rabbit anti-mouse IgG HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-358914; 1:5,000) and SuperSignal Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). The densities of bands were analysed using Image Lab software.

Taurocholate acids and diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) were purchased from Sigma‒ Aldrich (St. Louis, MO, USA). Recombinant human TGF-β, Y27632, U0126 and SB203580 were obtained from MCE Med-ChemExpress (Monmouth Junction, NJ, USA). JTE-013 and CAY1044 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The α-SMA, collagen 1, p-YAP (Ser127), and YAP antibodies were obtained from Cell Signaling Technology (Danfoss, MA, USA). β-actin, S1PR2 and Lamin B1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-linked anti-mouse and anti-rabbit IgG antibodies were obtained from Beyotime Biotechnology (Nanjing, China).

Reverse-transcription PCR (RT-PCR) was performed as previously described (1 (link)). Total RNA was extracted form MM cells, HUVECs, and primary patient samples using the RNA queous^®−4PCR kit (Life Technologies Japan, Ltd.). The RNA concentration was determined spectrophotometrically. Next, 82 ng RNA was used to synthesize cDNA using a first-strand cDNA synthesis kit (OriGene Technologies) under the following reaction conditions: 1 cycle at 22°C for 5 min, 1 cycle at 42°C for 30 min, and then1 cycle at 85°C for 5 min, followed by a hold at 4°C. RT-PCR was performed using a PCR Master Mix (Promega Corporation) and the Roche Light Cyber 2.0 detection system (Roche Diagnosis Gmbh). Thermocycling conditions were as follows: 95°C for 5 min, then 40 cycles at 95°C for 30 sec, 55.5°C for 30 sec, and 72°C for 1 min. The primer sequences were as follows: GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and GAPDH reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. The GAPDH primer was purchased from Life Technology Japan, Ltd. The specific PCR primers of S1PR1, S1PR2, S1PR3, S1PR4, S1PR5, SK1, and SK2 were purchased from Santa Cruz Biotechnology. The information of the sequences of these primers could not be provided from the company then the sequences are not publicly available.