Cell culture

Human breast cancer cell lines MCF-7 and MDA-MB-231 and murine melanoma cancer cell line B16F10 were obtained from NCCS, Pune, and were grown and maintained in required medium-like Minimal Essential Medium (GIBCO), Dulbecco’s Modified Eagle’s Medium (GIBCO), and McCoys’ 5A Medium (GIBCO) containing 10% fetal bovine serum (LifeTech, BioWhittaker) in a CO2 incubator at 37 °C.

Zymography

For gelatin zymography, gelatin (Sigma), Triton-X (Promega), NaCl, CaCl2, gelatin-Sepharose 4B beads (Amersham) were used. The method was followed according to the protocol by Moulik et al. [14].

Immunoprecipitation and Western blotting

Protein extraction from cells and immunoprecipitation assay was done following the protocol of Moulik et al. [14]. The protein content was estimated by Lowry’s method. Equal amount of protein was taken and immunoprecipitated from the supernatant using required primary antibodies and protein-G agarose beads and shaking them overnight at 4 °C. The resultant immune complex was thoroughly washed thrice in PBS and then subjected to Western blotting.

For Western blotting, the materials used were as follows: acrylamide (Promega), bis-acrylamide (Sigma), Tris (Promega), sodium dodecyl sulfate (Biogene), ammonium persulfate (LifeTech), glycine and stain (Coomassie Brilliant Blue), bromophenol blue, methanol, acetic acid, and TEMED for SDS page. Primary antibody used was anti-phospho-ERK, anti-ERK, anti-MMP2, anti-MMP9, anti-JNK, anti-phospho-JNK, anti-PI-3 K, anti-STAT-3 anti-integrins antibody, or as required (Sigma, Promega, Santa Cruz) and 2nd antibody both monoclonal and polyclonal (Promega/Santa Cruz) and substrate used was NBT/BCIP or Femto substrate for ECL (Pierce) NaCl, Tris, Tween-20, Tris, glycine, methanol, BSA, and nitrocellulose membrane. The method was followed according to Moulik et al. [14].

Cell invasion assay

For the assay, Millicell inserts (Millipore) and Matrigel (BD Biosciences) were used. The protocol was followed according to Moulik et al. [14]. Briefly, 24-well transwell plate (Corning) with 12 inserts were taken, and the lower chamber of each well was poured with 600-ml MEM SFCM. Control and fibronectin (Fn)-treated cells (100,000 cells/insert) were seeded in triplicate on membrane in the upper chamber of the insert. Cells were then allowed to grow for 24 and 48 h. After 24 and 48 h of incubation, media was pipetted out from membrane. SFCMs from lower chambers were collected and centrifuged at 3000 r.p.m for 3 min. The membranes of the inserts were washed thrice with PBS. Cells were then fixed with 4% formaldehyde solution, followed by washing with PBS. Cells were then stained with Gill’s hematoxylin for 10 min. Membranes were then washed thoroughly in running water. The upper side of the membranes was scraped with buds; membranes were then cut and mounted with glycerol. The cells migrated through the membrane pore were observed under microscope.

Wound healing assay

Cells were grown as a monolayer on culture plates in the absence (C) and in presence of 50 µg/ml theaflavin at 37 °C for 24 h (E). The monolayer was scratched with a sterile pipette tip, followed by washing with serum-free complete medium (SFCM) to remove cellular debris. Cell migration across the wound was observed by microscope and documented by photographs at 0 h, 6 h, 24 h, and 48 h [14].

siRNA transfectionSingle transfection procedure

Cells were plated 24 h prior to transfection. The cells were incubated at 37 °C for 24 h under 5% CO2. After incubation, the plate was washed with PBS (once) and layer with 1.75-ml Opti-MEM. The following mix was prepared and incubated at room temperature for 5 min.

Tube 1: siRNA: 100 nM (stock conc.: is 100 µM, siRNA: 2 µl) and Opti-MEM: 198 µl

Tube 2: Oligofectamine: 4 µl and Opti-MEM: 46 µl

The content of both the tubes was mixed and incubated for further 30 min. siRNA-lipid mix on the cells (dropwise with constant swirling) was layered. The plate was incubated at 37 °C for 4 h. Fresh media was added after 4-h incubation and incubate further 24 h. Cells were analyzed depending upon the experiment.

Double transfection (combinatorial transfection) procedure involves two rounds of siRNA transfection, and second round of transfection was done in the similar manner as described above after 24 h of the first round. Scramble control for siRNA was similarly prepared.

Constitution of siRNA

1 × nuclease-free siRNA buffer was prepared from 5 × provided by Dharmacon (use nuclease-free water, also provided by Santa Cruz). For 100-nM siRNA, 1-ml 1 × nuclease-free siRNA buffer was used. Aliquots the siRNA were stored at − 20 °C.