Fr180204

Differentiated CB-1 cells were preincubated with P38, ERK, and PKA signal inhibitor at 37 °C for 30 min and treated with 100 nM of CXCL12 for 2 h. SB203580 (#5633; Cell Signalling Technology) as a P38 inhibitor, FR180204 (#0320; Sigma-Aldrich) as an ERK inhibitor, and H89 (#9844; Cell Signalling Technology) as a PKA inhibitor were adjusted to a final concentration of 10, 25, and 10 μM, respectively. At the end of the reaction, cell lysate was harvested using RLT buffer and RNeasy Mini kit. Extracted RNA was reverse transcribed to cDNA and examined with qRT-PCR to detect UCP-1 mRNA expression.

Ang II, ET-1, PD123319, gallein (G_βγ inhibitor), and BQ788 were obtained from Tocris Bioscience (Ellisville, MO, USA). Recombinant human TGF-β1, valsartan, bosentan, ambrisentan, LY2109761, FR180204, and SB203580 were obtained from Sigma Aldrich (Saint Louis, MO, USA). SIS3 (Smad3 inhibitor) and FR900359 (G_αq inhibitor) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Fibroblast growth medium and related cell culture reagents were obtained from Promocell (Heidelberg, Germany).

PAR4-AP (AYPGKF-NH2) and corresponding scrambled peptide (YAPGKF-NH2) were from Peptides International, Inc. (Louisville, KY, USA). Disulfide HMGB1 peptides were from HMGBiontech (Milano, Italy). N-Acetylcysteine amide was purchased from Tocris (Minneapolis, MN, USA). FR180204 was from Sigma-Aldrich. H&E staining reagents were from Fisher Scientific. The rest of the materials used were from Sigma-Aldrich or as described in the methods.

Canine recombinant IL-1β and NGAL were purchased from Kingfisher Biotech, Inc. (Saint Paul, MN) and United States Biological (Salem, MA), respectively. TRIzol, anti-zonaoccludin-1 (ZO-1) mouse monoclonal antibody (Clone: ZO-1-1A12), Alexa Fluor 488-conjugated F(ab′)2 fragments of goat anti-rabbit IgG (H+L), Alexa Fluor 594-conjugated F(ab′)2 fragments of goat anti-mouse IgG (H+L), TO-PRO-3-iodide, and ProLong Gold Antifade Reagent were purchased from Life Technologies Co. (Carlsbad, CA). PrimeScript RT Master Mix and SYBR Premix Ex Taq II were obtained from TaKaRa Bio Inc. (Shiga, Japan). Rabbit monoclonal antibodies against E-cadherin (Clone: 24E10) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). The mitogen-activated protein kinase (MAPK) inhibitors FR180204, SB239063, SP600125, and U0126, and the IκB kinase inhibitors BAY-117082 and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) were purchased from Sigma-Aldrich Inc. (St Louis, MO). The NGAL assay kit was purchased from BioPorto Diagnostics A/S (Hellerup, Denmark). StatMate IV was purchased from ATMS (Tokyo, Japan). Culture plates, dishes, and flasks were obtained from Thermo Fisher Scientific, Inc. (St. Waltham, MA).

The MAPK inhibitors FR180204 (ERK1/2 MAPK; 30 mg/kg body weight i.v.), SB202580 (p38 MAPK; 30 mg/kg body weight i.v.), or SP600125 (JNK MAPK; 30 mg/kg body weight i.v.; Sigma Aldrich GmbH, Taufkirchen, Germany) were used to characterize the functional relevance of MAPK for uPA‐PAI‐1‐elicited neutrophil trafficking. A NE inhibitor (sivelestat; 150 µM; Sigma Aldrich) was used to evaluate the role of NE for 4T1 tumor cell proliferation. The competitive small‐molecule WX‐340 (10 mg/kg body weight i.p. for invivo experiments; Heidelberg Pharma AG, Ladenburg, Germany) was used to inhibit heteromerization of uPA and PAI‐1.

293 and 231 cells were transfected with same amount of pUK21-BikDD or pUK21-BikDDA. 16 hours later, cells were treated with pharmacological inhibitors as indicated and the harvested cells were lysed. Cell lysates were subjected to SDS–PAGE and protein bands were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, MA, USA). Primary Antibodies against Bik (sc-10770, Santa Cruz, CA, USA), β-actin (sc-47778, Santa Cruz, CA, USA), pospho-ERK1/2(#9106, Cell signaling technology, USA) and ERK1/2(#9102, Cell signaling technology, USA) and HRP-conjugated secondary antibodies (ab6721, ab6728; Abcam) were used. The immunoreactive bands were visualized with a chemiluminescent substrate (Santa Cruz, CA, USA). The relative protein expressions were analyzed by Gel pro software.
Pharmacological inhibitors: UO126 (5 μM, Beyotime, China), FR180204 (0.6 μM, Sigma, St. Louis, MO, USA), MG132 (10 μM, Beyotime, China) and cycloheximide (1 mM, Sigma, St. Louis, MO, USA) were utilized.

PAR4-AP (AYPGKF-NH2; to elicit BHA) and corresponding scrambled peptide (YAPGKF-NH2; as control) were from Peptides International, Inc. (Louisville, KY). N-Acetylcysteine amide was purchased from Tocris (Minneapolis, MN). FR180204, ethyl pyruvate and methyl cellulose were from Sigma-Aldrich. H&E staining reagents were from Fisher Scientific. Mouse HMGB1 enzyme-linked immunosorbent assay (ELISA, MBS2701751) kits were obtained from MyBioSource, Inc. (San Diego, CA). The rest of the materials used were from Sigma-Aldrich or as described in the methods.