Pf 562 271

Total cellular RNA was isolated using rna-OLS (OMNI Life Science, Hamburg, Germany) and reverse transcribed (Advantage First strand cDNA synthesis kit; Fermentas, St. Leon-Rot, Germany). qPCR and melting curve analysis were performed using Qiagen QuantiTect SYBR Green PCR kit in a LightCycler (Roche, IN). Expression of additional mRNAs was analyzed using the following TaqMan probes on an ABI PRISM 7300 Sequence Detection System, as described previously (42 (link)): 18S rRNA Hs99999901_s1, KLF4 Hs00358836_m1, SDC1 Hs00174579_m1, HPSE Hs00180737_m1. The 2^−ΔΔCt method was used to determine relative gene transcript levels after normalization to 18S rRNA. PF-562271 (Sigma-Aldrich) was used for 24 h at 10 μg/ml in some experiments.

Primary RBMECs (CellBiologics; Cat No. RA-6023) were cultured in endothelial cell medium (CellBiologics) supplemented with 2% fetal bovine serum, endothelial cell growth factors, and antibiotics (CellBiologics; Cat No. M1266-Kit). Media was changed every 2 days until cells reached confluence. All experiments were conducted between passages 4–6. In each experiment, cultures exposed to TNF for 6 h (10 ng/ml; Thermo Fisher Scientific) were compared with PBS control conditions. The FAK inhibitor, PF-562,271 (FAK-I; 0.5–2.5 μM Sigma Aldrich, St. Louis, MO) was added to RBMECs for 1 h before TNF addition for 6 h.

Cytokine and inhibitors; purified recombinant human HGF was obtained from Peprotech. The c-Met receptor inhibitor (PHA-665752 ≤ 1 μM), MAPK pathway inhibitor (PD98059 ≤ 40 μM), Arp2/3 complex inhibitor (CK-666 ≤ 200 μM) and CDC42 inhibitor (ML-141 ≤ 20 μM) were obtained from Cayman Chemical. The ROCK pathway inhibitor (Y27632 ≤ 7.5 μM) and FAK inhibitor (PF-562,271; 1 μM) were purchased from Merck. The PI3K pathway inhibitor (LY294002 ≤ 100 μM) and the Rac1 inhibitor (6-Thio-GTP≤50 μM) were obtained from Selleck Chemicals and Jena Bioscience, respectively. The functionally blocking β1-integrin antibody (≤10 μg/ml) and its mouse IgG1 isotype were purchased from Abcam.
Immunofluorescence; primary and secondary antibodies, rabbit anti-Met, and rat anti-α-tubulin were obtained from Invitrogen. Mouse anti-paxillin was obtained from BD Bioscience. Mouse anti–β1-integrin was obtained from Abcam. Alexafluor-conjugated secondary antibodies (488, 568, and 647 nm), Rhodamine phalloidin, DiO’, and DiOC₁₈(3) (3,3′-Dioctadecyloxacarbocyanine Perchlorate) were all purchased from Invitrogen. The mitochondrial staining kit, red fluorescence-cytopainter, was purchased from Abcam. Vectashield mounting medium was obtained from Vector Laboratories Ltd., and DAPI was obtained from Invitrogen.