Anti integrin β3

Anti-biglycan antibody (Abcam, ab49701 [39 (link)]), anti-integrin-β1 Antibody (clone: EP1041Y) (Abcam, ab52971 [40 (link)]), anti-Integrin-α5 (Abcam, ab150361) [41 (link)], integrin-β4 (Santa Cruz Biotechnology, INC, sc 9090 [42 (link)]), anti-Integrin-β3 (clone: D7×3P, Cell Signaling Technology, 13166 [43 (link)]). Dilutions of the antibodies are listed in the Supplementary methods.

NaHS was administered instead of H₂S. NaHS and the Pyk2 inhibitor (PF431396) were obtained from Sigma-Aldrich; The FAK specific inhibitor (PF573228), the Src inhibitor (PP2), and the Rac1 inhibitor (NSC23766) were purchased from Selleck Chemicals. CES-siRNA (sc-142618) was obtained from Santa Cruz. Rhodamine was obtain from Cytoskeleton, and DAPI from Beyotime; Following antibodies were used: anti-Rac, anti-Fak, anti-p-FAK³⁹⁷, anti-p-FAK⁹²⁵, anti-Src, anti-p-Src, anti-β-actin, anti-Integrin β1, anti-Integrin β3, anti-Cavenolin-1,anti-p-Pyk2 (Cell Signaling); anti-CSE (Santa Cruz); anti-Integrin β1-FITC, anti-Galectin-3 (eBbioscience); anti-CD68 (Biolegend); Lactate dehydrogenase (LDH) assay from BeyotimeInstitute of Biotechnology.

Anti-p53, anti-p21^waf1, anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1β, anti-GSK-3β, anti-Rb, anti-p27^kip1, anti-XIAP, anti-bax, and anti-integrin β1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p) Akt Serine473, anti-pGSK-3β Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin β3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-β-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin β2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals.

We performed IHC of tumor biopsies from six patients with advanced NSCLC bearing EGFR mutations, before erlotinib treatment and after erlotinib resistance. The protocol was approved (approval no. 2018-G-106) by the Institutional Medical Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University and was in agreement with the Declaration of Helsinki. Written informed consent was obtained from the patients prior to study commencement. Detailed patients’ data are summarized in Supplemental Table S1. After the process of routine deparaffinization, rehydration and antigen retrieval, the tissue slides were incubated with primary anti-integrin β3 (Cell Signaling Technology, Danvers, MA, USA, Cat #13166, RRID: AB_2798136, 1:250) and anti-AXL (Cell Signaling Technology, Cat #8861, RRID: AB_10998619, 1:300) monoclonal antibodies at 4 °C overnight. The next day, horseradish peroxidase-conjugated antibodies and the diaminobenzidine peroxidase substrate were utilized for the establishment of staining.

BMMs on coverslip were fixed by 4% paraformaldehyde for 10 min at RT. After three washings with PBS, the cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 min at RT. After another three washings with PBS, cells were blocked with 5% goat serum for 1 h at RT. Following another three washings with PBS, the cells were incubated with anti-integrin β3 (1:100, Cell Signaling Technology) or anti-paxillin (1:60, Santa Cruz Biotechnology Inc.) with 1% BSA overnight at 4 °C. Cells were washed again with PBS, and incubated with an Alexa fluor 488 labeled secondary antibody (1:400, Fisher Scientific, Suwanee, GA, USA) with 1% BSA for 1 h at RT. After washing with PBS, cells were incubated with rhodamine phalloidin (1:140, Cytoskeleton, Inc., Denver, CO, USA) for 1 h at RT. Cells were washed again with PBS and mounted on a slide with Prolong Gold anti-fade reagent with DAPI (Fisher Scientific) overnight at RT. Digital images were recorded by an Olympus BX61fluorescent microscope. Fluorescence intensity analysis was performed using 20 cells per image. Fluorescence intensity was quantified by Adobe Photoshop using the following formula: corrected cell fluorescence = integrated density − (area of selected cell × mean fluorescence of background).

Immunoblotting was conducted as reported previously. Briefly, cell samples were lysed in lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; and the protease inhibitor, 1 mM PMSF), and tissue samples were prepared in tissue protein extraction reagent (Thermo Fisher Scientific). Protein (40 μg) was processed for the analysis. The antibodies used for the analysis were as follows: anti- Integrin α5 (1:200, Santa Cruz Biotechnology), anti-Integrin β3 (1:1000, Cell Signaling Technology), anti-SP1 (1:1000, Proteintech), anti-p-MEK1/2 (Ser218/222, 1:1000, Cell Signaling Technology), anti-MEK1/2 (1:1000, Cell Signaling Technology), anti-p-NEDD4L (Ser448, 1:1000, Abcam), anti-NEDD4L (1:1000, Proteintech), anti-ubiquitin (1:500, Santa Cruz Biotechnology), anti-GAPDH (1:1000, Beyotime).