Bovine serum albumin (bsa)

Cells were grown in a chambered glass slide (Ibidi, Germany) and treated with 10 μM BAPTA-AM (Sigma-Aldrich, USA) for 1 h. Following this, 200 μl collagen I mix [collagen R-1.8 mg/ml (Serva, Germany), 5X DMEM (Himedia, India)—200 μl per ml of collagen I mix, NaOH (Alfa Aesar, USA)—8 ul per ml of collagen I mix] was added to each well and thermally gelled at 37 °C for 2–3 h. Collagen gel was layered with 100 μl of media with 20% FBS and incubated for 48 h. After incubation, media was aspirated, gels were washed with PBS, fixed with 3% paraformaldehyde (Sigma-Aldrich, USA) (15mins), permeabilized with 0.5% Triton X-100 (Alfa Aesar, USA) (30 min), and blocked with 1% BSA (Himedia, India) (30 min). Cells were then stained with Phalloidin-TRITC (Sigma-Aldrich, USA) (for 90 min) and Hoechst (Himedia, India) (for 15 min) at room temperature. Invaded cells were imaged using laser scanning confocal microscope with × 63 oil immersion objective (Leica Microsystems, Germany). Confocal Z slices were collected for each well at 40 μm from the bottom, and sequential Z slices were used to construct the 3D images (Yang and Yang 2013 ).

S2R+ cells were individually transfected with pUAST-attB-mCherry::Spo20, pJFRC-LNS2(RDGB)::GFP and pJFRC-LNS2(RDGB-D1164A)::GFP for 48 h, following which cells were lysed with Protein Lysis Buffer (50 mM Tris-Cl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 0.27 M Sucrose, 0.1% β-Mercaptoethanol). In parallel, strips made using nitrocellulose membranes [Hybond-C Extra; (GE Healthcare, Buckinghamshire, UK)] were spotted with increasing picomoles of DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, Avanti Polar Lipids, 840875). The spotted membrane was dried and then blocked using 5% BSA (HiMedia) in TBST for 2 h at RT. Following this, the strips were incubated overnight at 4°C with the remaining cell lysate. Next the membranes were washed extensively five times with 0.1% TBST and then incubated with anti mCherry (Thermo Fisher Scientific PA5-34974, 1:4000) or anti-GFP antibody [(sc-9996), 1:2000] at RT for 2 h. The membranes were then probed with the appropriate HRP-conjugated secondary antibody (Jackson Immunochemicals; 1:10,000) and binding was detected using ECL (GE Healthcare) in a LAS4000 instrument.

ZnONPs was purchased from Sigma aldrich (cat. no.677450 and 544906) and FA (C₁₀H₁₀0₄, 98%) was obtained from Sisco research laboratories Pvt Ltd. RPMI 1640, MTT, BSA, acridine orange and ethidium bromide were obtained from Himedia.

Tetraethyl thiuram (TTD), aristolochic acid (AA), silymarin (SLN), phorbol 12-myristate 13-acetate (PMA), porcine skin gelatin, collagen-I/IV, laminin, fibronectin, Hoechst stain, DNase 1, ficoll-paque, dextran and phosphatase inhibitor cocktail were obtained from Sigma-Aldrich (Bangalore, India). BSA, ethanol, dimethyl sulfoxide (DMSO; HPLC grade), Tween-20 and Hank’s balanced salt solution (HBSS) were purchased from HiMediaLaboratories, Pvt. Ltd. (Mumbai, India). SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA). U0126 (MEK 1/2 inhibitor), antibodies against p-ERK, β-actin and cell lysis buffer were purchased from Cell Signaling Technology (Massachusetts, USA). HRP tagged anti-rabbit IgG and anti-mouse IgG were procured from Jackson ImmunoResearch (Philadelphia, USA). The rabbit polyclonal anti-citH3, rabbit polyclonal anti-H3, mouse monoclonal anti-myeloperoxidase (anti-MPO) and anti-PAD4 were obtained from Abcam (Cambridge, UK). All other chemicals and reagents used in this study are analytical grade.

Whole cell lysates were obtained by resuspension of cell pellets in 500 μl RIPA buffer (Sigma, USA) and 40 μl of protease inhibitor cocktail (Sigma, USA). The lysates were estimated for protein concentration using BCA Assay method. Protein extracts (40 μg) were subjected to SDS-PAGE using 12% Tris/HCl SDS gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Membrane Technologies, India). Membranes were blocked using 3% BSA (Himedia, India) for 2 hours, followed by incubation with primary antibody against β- actin (Abcam,UK, 1:500) and tyrosine hydroxylase (Santa Cruz, USA, 1:500) in 1% BSA- phosphate saline buffer (PBS) overnight at 4°C. Post incubation, membranes were washed thrice in TBST and incubated with the appropriate horseradish peroxidase (HRP) - conjugated secondary antibody (1/4,000) (Dako, USA) for 2 hours at room temperature. Membranes were developed with chemiluminescence detection reagents (Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).