5 fluorouracil

Lewis lung carcinoma (LLC1) cells were obtained from the American Type Culture Collection (ATCC, Baltimore, U.S.A.). Cultures were maintained in RPMI-1640 supplemented with 10% FCS. All experiments were carried out within 6 months of resuscitation. LLC1 cells were incubated with etoposide (400 μM), curcumin (160 μM), tamoxifen (40 μM), staurosporine (1 μM), cisplatin (400 μM) or 5-fluorouracil (160 μM) (all from Enzo Life Sciences) for 24 hrs to induce apoptosis. Apoptotic bodies were purified as described by Fransen et al., 2009 (28 (link)); briefly, cells were centrifuged at 1500 g for 10 mins at 20°C. The supernatant was centrifuged at 15700 g for 50 mins at 20°C. Apoptotic bodies were resuspended in PBS.
4 x 10⁶ LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.

VitK3, N-ethylmaleimide (NEM), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), H₂O₂ and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D and 5-fluorouracil were from Enzo Life Sciences (Villeurbanne, France). Hygromycin B, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet, Vybrant Apoptosis Assay Kit #3, propidium iodide (PI), tetramethylrhodamine methyl ester (TMRM), 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS) were from Life Technologies (Eugene, OR, USA). The following antibodies were used: anti-PRX1 (AbFrontier, Seoul, Korea); anti-PRX3 (Abcam, Cambridge, MA, USA); anti-TRX2 (R&D Systems, Minneapolis, MN, USA); anti-NQO2 (Proteintech, Chicago, IL, USA); anti-GFP (Life Technologies); anti-β-actin (Santa Cruz Biotechnology); anti-α-tubulin (Sigma-Aldrich) and anti-TRX1 (Cell Signaling Technology, Danvers, MA, USA). ON-TARGETplus SMARTpool small interfering RNA (siRNA) targeting PRX1 (siPrx1), and non-targeting pool control siRNA (siCon) were from Dharmacon (Lafayette, CO, USA). Mammalian expression vectors encoding HyPer targeted to cytosol, nucleus and mitochondria were purchased from Evrogen (Moscow, Russia).

FTY720 was from Sigma-Aldrich (St. Louis, MO, USA). Okadaic acid was from Wako Pure Chemical (Tokyo, Japan). 5-Fluorouracil and doxorubicin were from Enzo Life Sciences (Farmingdale, NY, USA). All other reagents were of analytical grade and obtained from commercial sources.