Calpeptin

For transient transfection, the EGFP-tagged FAK, paxillin, vinculin, and talin plasmids were transfected into IGROV1 cells using Lipofectamine 3000 (Invitrogen, San Diego, CA) for 48 h. Thapsigargin, YM-58483, PD105606, PD151746, ALLN, and cisplatin were purchased from Sigma–Aldrich (Saint Louis, MO). Calpeptin and calpastatin were purchased from Cayman Chemical (Ann Arbor, MI). 2-APB, SKF-96365, and fura-2/AM were purchased from Invitrogen (San Diego, CA). SR-T100 was kindly provided by G&E Herbal Biotechnology (Tainan, Taiwan).

LEI-106, WIN-55,212, calpeptin, G06983, SR3677, 2-AG, 2-AG-d5, and AEA-d4 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Cells received LEI-106 (650 µM, 1.3 mM) dissolved in 0.9% DMSO in cell media 15 min prior to analysis. The doses of LEI-106 used for cell assays were derived as equimolar doses to previous in vivo assays, assuming 50% and 100% bioavailability. Titrated doses of LEI-106 in 0.9% DMSO (100 nM, 10 µM, 100 µM, 325 µM, 650 µM, 1.3 mM, 10 mM) were utilized to assess for cell viability. 2-AG was dissolved in 0.9% DMSO in cell media and was administered to cells in 600 pmol dose. calpeptin (10 µM), G06983 (100 nM), and SR3677 (100 nM) were applied 30 min before LEI-106 treatment.

Cells were pelleted by centrifugation (800 g for 3 min), resuspended in 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT, 0.1% Triton X-100, and lysed for 30 min at 4°C. A minimum of 10 μg total protein per reaction was diluted to a final volume of 100 μl with calpain activity assay reaction buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂ and 1 mM DTT). Duplicates for each condition were prepared of which one was treated with 100 μM calpain inhibitor calpeptin [CPT (Cayman Chemicals, 119591-20-5), 30 min at room temperature]. Calpain activity was measured as 360 nm and 460 nm (excitation/emission) in 2 mM CaCl₂, 5 mM cysteine and 50 μM CAPN substrate (N-succinyl-Leu-Leu-Val-Tyr 7-Amido-4-Methylcoumarin; ACM substrate, Sigma Aldrich, S6510) after 1 h incubation at 37°C in a 96-well plate and using an Infinite 2000 plate reader (Tecan). Emission values obtained following CPT treatment were subtracted from emission values obtained from untreated control samples to exclude the contribution of other proteases. Calpain activity was expressed relative to control condition or in total value as relative fluorescence units (RFUs) as indicated in the figure legends.