Saracatinib

Neutrophils were isolated from venous peripheral blood obtained from AAT competent (PiMM) and deficient (PiZZ) volunteers. Density gradient centrifugation was conducted in LymphoPrep (Axis-Shield PoC AS, Oslo, Norway) to isolate the red cell pellet containing the neutrophil population. Neutrophils were further purified as described previously^{12 (link)}. Neutrophils were also isolated from the bone marrow of mice as previously described^{6 (link)}. Male and female adult C57BL/6J and Pltp^−/− mice^{65 (link)}, were maintained in a specific pathogen-free facility at SUNY Downstate Medical Centre. Neutrophils were placed in PBS supplemented with 4 mM glucose prior to stimulation. Cells were treated with either 2 μM PiMM or PiZZ AAT, 0.5 μM PLTP, 100 nM Saracatinib (LC Laboratories) or 100 nM dasatinib for 10 minutes prior to LTB₄ or fMLP stimulus. Secreted protein and whole cell protein were collected at several time points. All animal experiments were performed with approval from SUNY Downstate’s Institutional Animal Care and Use Committee. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) guidelines and according to the Declaration of Helsinki conventions for the use and care of animals.

Antibodies against P-FAK (Y576/577) (#3281), P-Paxillin (Y118) (#2541), P-p130Cas (Y410) (#4011), PARP (#9542), Akt (#9272), Src (#2109), Active-Caspase-3 (#9664), P-Akt (S473) (#4051), Caspase 7 (D2Q3L) (#12827), VHL (#2738), MMP2 (#4022), MMP9 (#3852) and HSP90 (#4874) were from Cell Signaling Technologies (Danvers, MA). Antibodies against Paxillin (610052), FAK (610088) and p130Cas (610271) were from BD Biosciences (San Jose, CA). Antibody against P-Src (Y418) (LF-PA20465) was purchased from AbFrontier. Antibody against PCNA was from Abcam (Cambridge, UK). GAPDH (AM4300) was purchased from Life Technologies (Carlsbad, CA). Antibody against HA-12CA5 (11666606001) was from Roche. Dimethyl sulfoxide was from Sigma Aldrich (St Louis, MO). Saracatinib and GDC-0941 were obtained from LC Laboratories (Woburn, MA).

SP600125 was purchased from Beyotime Institute of Biotechnology and Sigma. JNK inhibitor VIII was purchased from Merck (Darmstadt, Germany). Saracatinib was purchased from LC Laboratories. Antibodies for p-IGF-1R (Y1135) (#2182-1) were purchased from Epitomics (Burlingame, CA, USA). Antibodies for IGF-1R (#3027), p-Akt (S473) (#4060), Akt (#3063), p-Erk1/2 (T202/Y204) (#4370S), Erk1/2 (#4695), p-Src (Y416) (#6943P), Src (#2123), p-JNK (T183/Y185) (#9251), JNK (#9258P) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for β-actin (#KM9001) was purchased from Tianjin Sungene Biotech Co., Ltd., Tianjin, China.

PTx was from Sigma (St. Louis, MO, USA). Trastuzumab was from Genetech (South San Francisco, CA, USA). Laptinib, GDC0941 and saracatinib were from LC Laboratories (Woburn, MA, USA). Antibodies for AKT (no. 4685), phospho-AKT^S473 (no. 4060), Src (no. 2109), and phospho-Src^Y416 (no. 6943) were from Cell Signaling Technology (Danver, MA, USA); GADPH (sc-47724) was from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for mouse FITC-conjugated CD24 (#101806), allophycocyanin-conjugated CD49f (#313616), and phycoerythrin-conjugated CD61 (#104308) were from Biolegend (San Diego, CA, USA). GCV was from TCI America (Portland, OR, USA).

OE19 cells were obtained from the European Collection of Cell Cultures (ECACC), and OE33 cells were purchased from the Sigma (St. Louis, MO). OE19 is ERBB2-amplified gastroesophageal cancer cell line and sensitive to lapatinib, hence OE33 is ERBB2- and MET-amplified gastroesophageal cancer cell line and has intrinsic resistance to lapatinib. OE19 and OE33 were cultured in a humidified, 5% CO₂ atmosphere at 37°C in Roswell Park Memorial Institute (RPMI-1640; GIBCO BRL, Grand Island, NY) medium supplemented with 10% fetal bovine serum. Tyrosine kinase inhibitors (lapatinib, saracatinib, and crizotinib) were purchased from the LC laboratories and were dissolved in dimethylsulfoxide (DMSO). Trastuzumab was purchased from the Department of Pharmacy at the Dana-Farber Cancer Institute.

LPA and PTx were from MilliporeSigma. Human and mouse SDF1-α were from Pepro Tech. EGF was from Gold Biotechnology. Sapitinib, erlotinib, and cp-724714 were from Shelleck Chemicals. Trastuzumab was from Genetech. Lapatinib, GDC0941, and saracatinib were from LC Laboratories. Antibodies for EGFR (no. 2232), phospho-EGFR^Y1068 (no. 3777), HER2 (no. 2165), phospho-HER2^Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT^S473 (no. 4060), Src (no. 2109), phospho-Src^Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2^T202/Y204 (no. 4370) were from Cell Signaling Technology. GAPDH (sc-47724) was from Santa Cruz Biotechnology. Ki67 (GTX16667) was from GeneTex. EGFR (no. 4267) from Cell Signaling Technology and phospho-Src^Y418 (ab4816) from Abcam were used in immunohistochemical staining. Antibodies for LPAR1 (NBP1-03363SS), CXCR4 (NB100- 56437SS), and CXCR7 (NBP2-24779SS) were from Novus Biologicals.