Tnf α

matrigel invasion assay was performed according to the science advisory board website (www.scienceboard. net/resources/protocols). In detail, the matrigel (BD Bioscience, UK) was thawed on ice overnight and diluted to 1 mg/ml in cold serum-free Ham-F12 (Gibco, USA). A 100 μl of matrigel was applied into chamber of 24-well transwell (Corning Incoporated, USA) and incubated at 37°C for 4-5 h for gelling. Next, 100 µl of 1x 106 cells/ml KKU-100 was seeded into the transwell and subsequently TNF-α (Prospec, USA) was added at a final concentration of 0.1, 1, 10, and 100 ng/ml. The lower chamber of the transwell was filled with 650 μl of Ham-F12 (Gibco, USA) containing 1% FBS and 1X penicillin/streptomycin (Biowest, France) and subsequently incubated at 37°C for 24 h. For detection, cells were fixed with 3.7% formaldehyde in 1X PBS for 2 min and then permeabilized by 100% methanol at room temperature for 20 minutes. The transwell was washed with 1X PBS twice and stained with Giemsa (Sigma, USA) for 15 minutes. After washing with 1X PBS, non-invaded cells on the top of the transwell was removed by a cotton swab and invading cells were counted under a light microscope (Olympus, USA). Time-course analysis was performed by choosing optimal concentration (10 ng/ml) of TNF-α (Prospec, USA) and the number of invading cells were counted at different time points; 0, 6, 12, 24 and 48 h.

Penicillin and streptomycin (Pen/Strep), Hanks' solution, DMEM, CMRL 1066 medium and trypsin-EDTA were obtained from Biological Industries (Bait Haemek, Israel) . FBS was from Hyclone Laboratories (Logan, UT, USA). Protein-G Agarose beads and antiactin antibodies were from Santa Cruz Biotechnology. Insulin, collagenase (type XI), protease inhibitor cocktail and 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) were from Sigma Chemicals. Enzolyte-caspase RH110 Caspase3/7 Assay kit was from AnaSpec Ltd (San Jose, CA, USA). zVADfmk was from MegaPharm Ltd (Raanana, Israel). Mouse insulin ELISA kit was from Mercodia (Uppsala, Sweden). CellTiter-Blue assay kit was from Promega (purchased through Biological Industries, Beit HaEmek, Israel). Monoclonal p-Tyr (PY-20) antibodies were from BD Biosciences (San Jose, CA, USA). Anti MCL-1 antibodies (Ab 32087) were purchased from Abcom (London, UK). Cytokines IL-1β and IFN-γ were purchased from MD Biosciences (Ness Ziona, Israel). TNF-α was provided by Prospec-Tany Technogene (Rehovot, Israel) . Cytokine mixture (1x-cytomix) consisted of 3 nM TNF-α, 3 nM IFN-γ and 1.5 nM IL-1β (biological activity: 10 U/ng (TNF-α, IFN-γ) and 200 U/ng (IL-1β)). Human insulin ELISA kits was purchased from Mercodia (Uppsala, Sweden).