Pd184352

U0126, PD0325901, PD184352, PD98059, KU0063794, Deforolimus, Perifosine, LY294002, GSK429286A, Y-27632 MG-132 and rapamycin were purchased from Selleck Chemicals. FTI-277, U73122, Vinblastine (VBT), Latrunculin B (LB) and Dynasore (Dyn) were purchased from Sigma. PDGF-AA and FGF2 were purchased from PeproTech.

The Met polyclonal antibody, kindly provided by Dr. Morag Park (McGill University, Montreal, QC, Canada), was raised against an epitope in the C-terminal region of human Met, distinct from those altered in the variants (Additional file 1) [8 (link),21 (link)]. The Phospho-Tyr (p-Tyr100), phospho-Akt (Ser473), and phospho-Erk1/2 (p44/42MAPK, Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The pan-Shc and phospho-Tyr Shc (Tyr239/240) antibodies, that recognize the p66, p52, and p46 isoforms of ShcA, and the Erk2 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The α-tubulin and β-actin antibodies were from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). The Grb2 and E-cadherin antibodies were purchased from BD Transduction Labs (Lexington, KY, USA). The MEK1/2 and PI3K inhibitors, U0126 and LY294002, were purchased from Cell Signaling Technology, while the MEK1/2 inhibitors AZD6244 and PD184352 were obtained from Selleck Chemicals (Houston, TX, USA).

Succinimidyl ester conjugated to Alexa Fluor 647 and DAPI were purchased from Life Technologies (Burlington, ON). The following small molecule inhibitors were purchased from Selleckchem (Houston, TX): Rapamycin, Torin2, SB203580, VX-702, SB202190 (FHPI), BIRB 796 (Doramapimod), FR 180204, PD184352 (CI-1040), PD98059, JNK Inhibitor II, JNK Inhibitor IX. Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155). ON-TARGETplus SMARTpool siRNAs for the genes of interest as well non-targeting negative control siRNAs were obtained from Dharmacon (Lafayette, CO). Phospho-p38 MAPK (Thr180/Tyr182) antibody (RRID:AB_2139682) for immunofluorescence was purchased from Cell Signaling (Beverly, MA).

For nonspecific differentiation, ESCs were washed in -LIF medium and then seeded at the cell densities specified below in each assay, as well as passaged on day one after seeding. Cells were assayed at 24, 48, 60, and/or 72 hr according to the experiment. For neural differentiation, ESC were first maintained in NDiff 227 medium (N2B27, Clontech) supplemented with 3 μM CHIR99021 (Selleck Chemicals) and 1 μM PD184352 (Selleck Chemicals), defined as 2i, to promote self-renewal in the absence of serum and LIF. Differentiation occurred in N2B27 in the absence of 2i and assayed at 24, 48, and 72 hr. For definitive endoderm differentiation, ESCs were grown in DMEM supplemented with 1% FBS, PSG, and 5 μM IDE1 (Cayman Chemical Company) and assayed at 48 hr. For EpiLC differentiation, ESCs were grown in N2B27 supplemented with 20 ng/mL activin A (R and D Systems), 12 ng/mL bFGF (Thermo Fisher), and 1% KOSR (Thermo Fisher) as previously shown (Hayashi et al., 2011 (link)), and assayed at 72 hr. For verteporfin-related experiments, verteporfin was diluted in DMSO to a concentration of 1 mM and then further diluted in fresh media during media changes, and cells were protected from light with aluminum foil. HEK293T cells (ATCC CRL-3216) were cultured in DMEM supplemented with 10% FBS and PSG. All cells were grown at 37°C in the presence of 5% CO₂.

To block EV entry, EV were incubated with recombinant Annexin-V (2 μg/mL, BD Biosciences) for 30 minutes prior to addition to hTERT-HUVECs. To block virus entry EV were incubated with 50 μg/mL heparin (Lonza). To block autocrine IL-6 feedback, 10 ng/mL IL-6 receptor (sIL-6R, Peprotech #200-06R) was added to cells 24 hours before EV addition. To inhibit MEK and ERK1/2, AZD6244 (SelleckChem) and PD184352 (SelleckChem), respectively were used to treat cells 24 hours prior to addition EV addition at indicated concentrations.

Melanoma cell lines (details in Supporting Information) were grown in DMEM/10% FCS (PAA, Yeovil, UK). PD184352, AZD6244, U0126 and vemurafenib were from Selleck Chemicals (Newmarket, UK).