Epigallocatechin gallate (egcg)

Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor^® 568, CellTrace Calcein Red-Orange, AM and DiOC18³ were from Invitrogen.

EGCG was purchased from Tocris bioscience (Cat: 4524, Minneapolis, MN, USA) and was reconstituted in dimethyl sulfoxide (DMSO) and stored at −20 °C for in vitro experiments. For in vivo experiments, EGCG was either purchased from ENZO (Cat: ALX-270–263, Farmingdale, NY, USA) or isolated from crude green tea mixture and stored at −20 °C as powder. The isolation of EGCG was performed as follows. Two (2) grams of crude green tea mixture was dissolved in MeOH/DCM 50:50 mixture and applied to a silica gel column equilibrated with a MeOH:DCM (4:96, v/v) solvent mixture. The 4–7% solvent system was used to elute EGCG and with the 7% MeOH/DCM solvent. EGCG, was collected in 16 fractions (each 25 mL). Finally, 704 mg EGCG was obtained and was of >97% purity as confirmed via NMR and LC/MS-MS analyses. Ruxolitinib was purchased from Selleckchem (Cat: S1378, Houston, TX, USA) and was reconstituted in DMSO and stored at −20 °C.

Cells (1 × 10⁴) were seeded in a 96-well plate, incubated for 24 h, and then treated with various concentrations (10, 20, 40, and 80 μM) of EGCG (Enzo Life Sciences, Farmingdale, NY, Purity ≥98% by HPLC) in serum-free media for 24 h. The effect of EGCG on the viability of PC-3 cells was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) assay as described previously [39 (link),40 (link),41 (link)]. The absorbance at 570 nm was measured using a microplate reader (Sunrise RC, TECAN, Mannedorf, Switzerland). Cell viability was expressed as a percentage of the control.