Pf 562271

To mimic loss of attachment, cells were seeded in culture dishes coated with polyHEMA (Sigma-Aldrich) to prevent cell adherence for various period of time. The cell suspensions were placed into regular culture dishes 16 h prior to end-point viable cell counting. To identify the signaling pathways that promote survival of detached cells, a panel of chemical inhibitors were added in culture medium: PTK2/FAK inhibitor – 0.5 μM PF562271 (Selleckchem, Houston, TX, USA), ERK1/2 inhibitor – 10 μM U0126 (LC Laboratories, Woburn, MA, USA) or 10 μM PD98059 (Santa Cruz Biotechnology, Dallas, TX, USA), AKT inhibitor – 30 μM ZSTK474 (LC Laboratories), TGFBRI/II inhibitor – 10 μM LY210761 (Selleckchem), SRC inhibitor – 3 μM dasanitib (LC Laboratories) or Notch inhibitor – 5 μM DAPT (Selleckchem).

Human primary pancreatic CAFs were cultured in DMEM/F12 0.5% FBS overnight before being treated with new DMEM/F12 0.5% FBS ± FAK inhibitor (PF‐562271, Selleckchem, #S2890) at 1 μM for 48 or 72 h. Mouse tumour cells were cultivated a week with 45% DMEM/F12, 45% DMEM and 10% FBS, then a week with DMEM/F12 10% FBS, and finally, cultured in serum‐free DMEM/F12 for 48 h. CM from either CAFs or tumour cells were then harvested and filtered through 0.22‐μm filter (Millipore; SLGS033SS).

Anti-α-tubulin and GAPDH antibodies were from Sigma-Aldrich. Antibodies against human EpCAM, total ERK and Thr202/Tyr204-phosphorylated ERK, total AKT, Ser473-phosphorylated AKT, total HGFR, Tyr1234/1235-phosphorylated HGFR, Non-phospho (Active) β-Catenin (Ser45), β-Catenin, E-cadherin, Vimentin, Snail, Slug, and Twist were from Cell Signaling Technology. LY294002 (AKT inhibitor) was also from Cell Signaling Technology. SU11274 (HGFR inhibitor), Crizotinib (HGFR inhibitor), U0126 (MEK inhibitor), PF-562271 (FAK inhibitor), TAPI-1 (ADAM17 inhibitor), DAPT (γ-secretase inhibitor), and BIO (GSK3 beta inhibitor) were obtained from Selleck Chemicals. Crizotinib (HGFR inhibitor) was obtained from Med Chem Express. Antibodies against total GSK3 beta, phosphorylated GSK3 beta (phospho S9), phosphorylated ADAM17 (phospho T735), total ADAM17, phosphorylated presenilin 2/AD5 (phospho S327), total presenilin 2/AD5, V5-tag, 6× His-tag, and c-Myc-tag, as well as the Met (pY1234/pY1235) + total Met ELISA Kit (ab126451) were obtained from Abcam. Human HGFR (c-MET) and HGF recombinant proteins were obtained from Sino Biological. The four mutant Snail constructs were obtained from Addgene.

ART was purchased from Guilin Pharmaceutical (Shanghai, China), dissolved in dimethyl sulfoxide (DMSO) and stored at − 80 °C. The concentration of DMSO in all experiments was 0.1%. The FAK inhibitor PF562271 was purchased from Selleck Chemicals (Houston, TX, USA). CCK-8 was obtained from BioSharp (Wuhan, China). The PE-Annexin V/7-AAD kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). TRIzol reagent was purchased form Invitrogen/Thermo Fisher Scientific (Carlsbad, CA, USA). Prime Script RT reagent Kit and SYBR® Premix Ex Taq™ were purchased from Takara (Tokyo, Japan). The BCA protein assay kit was from Pierce/Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against FAK (# 3285), p-FAK (# 3283), Akt (# 4691), p-Akt (# 4060), GSK-3β (# 9315), p-GSK-3β (# 9336), β-catenin (# 9562), Bax (# 2772), Bcl-2 (# 3498) and GAPDH (# 2118) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-rabbit secondary antibody IgG H + L (# 5151) was purchased from Cell Signaling Technology Inc.

hMSCs were seeded into four-well plates and exposed to adipogenic induction media composed of DMEM, 10% fetal bovine serum (FBS), 10% horse serum (Gibco, U.S.A.), 100 µM dexamethasone (Sigma, U.K.), 1 µM Rosiglitazone (BRL) (Novo Nordisk Bagsvaerd, Denmark), 3 µg/ml insulin (Sigma, U.K.), 450 µM isobutylmethylxanthine (IBMX) (Sigma, U.K.), and 1% penicillin-streptomycin (Sigma, U.K.) supplemented with FAK inhibitors (PF-573228 and PF-562271) or IGF-1R/InsR inhibitors (NVP-AEW541 and GSK1904529A), which were purchased from Selleckchem Inc. (Selleckchem Inc., Houston, TX, U.S.A.). Inhibitors were used at 5 µM throughout all experiments. Adipocyte induction medium (AIM) was changed every 2 days and for 7 days. Previous published work from our group indicated day 7 as a time point on which adipogenic markers were up-regulated significantly in an enriched population of 70–80% of adipogenic populations [8 (link)].

Cell viability was estimated by CCK-8 assay kit (5007, Japan) according to the manufacturer’s instructions. Absorbances were read by microplate reader (ELX800, USA) at 490 nm. FAK inhibitor (PF-562271) and AKT inhibitor (MK-2206 2HCl) were purchased from Selleck, China. Cell viabilities were expressed as percentages according to the following formula: (%) = A490 (sample)/A490 (control) × 100.

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].