Z devd fmk

The cells were seeded into 24-well plates at 2 × 10⁴ cells/well and were grown overnight and then pre-treated with inhibitors of caspase-3 (Z-DEVD-FMK, 50 µM, BioVision, San Francisco, CA, USA), caspase-8 (Z-IETD-FMK; 50 µM, San Francisco, CA, USA), and caspase-9 (Z-LEHD-FMK; 50 µM, San Francisco, CA, USA) for 2 h. Cells were then treated with 5 μM Lobocrassin B for 24 h. The cell viability were examined by MTT assay.

Recombinant SATB2 protein (250–500 ng; Abnova) and recombinant active caspase 3 (0.5 μg; Chemicon, Thermo Fisher Scientific, Waltham, MA, USA) or recombinant active caspase 7 (0.5 μg; BioVision, Milpitas, CA, USA) were incubated for 3 h in a cleavage assay buffer (50 mM Hepes, pH 7.5, 0.1 M NaCl, 10% (v/v) glycerol, 0.1% Chaps, 10 mM dithiothreitol) containing either dimethyl sulphoxide (DMSO) or z.DEVD.fmk (20 μM; BioVision; [36 (link),37 (link)]), as indicated. Reactions were stopped by the addition of the Laemmli sample buffer and subjected to SDS-PAGE. Mass spectrometry was performed at the Ottawa Hospital Research Institute Proteomics Core Facility (Ottawa, Canada). MASCOT 2.3.01 software (Matrix Science, Boston, MA, USA) was used to infer peptide and protein identities from the mass spectra.

The Caspase 3/7 activity assay was used to measure apoptotic death and performed mainly as described previously [24] (link). Briefly, the cells were seeded in 24 well plates and reverse-transfected with pre-miRs or siRNAs for 48 hours (Table S2 in File S1). The Caspase 3/7 inhibitor (z-DEVD-fmk) (Biovision, San Francisco, CA, USA) was added 6 hours post-transfection (final concentration 25 µM). Caspase 3/7 activity in cell lysates, measured by the liberation of AFC (excitation, 400 nm; emission 489 nm) from the substrate Ac-DEVD-AFC (Biomol, Plymouth Meeting, PA, USA), was measured using a multiplate reader; Multiscan MCC/340 (ThermoFisher Scientific).

Caspase inhibitors were used to block caspase expression. Briefly, HepG2 cells were induced by 3.0 mmol/l VPA combined with 40 μmol/l caspase 3 inhibitor Z-DEVD-FMK, caspase 8 inhibitor Z-IETD-FMK and caspase 9 inhibitor Z-LEHD-FMK, respectively (BioVision, Inc.). Apoptosis was detected using the aforementioned method following 48 h. Cells induced by VPA alone were used as the positive control, and normal control cells were cultured with culture medium.

Glutathione monoethyl ester (GSH-MEE) was purchased from Calbiochem (La Jolla, CA, USA). Z-DEVD-FMK and Z-VAD-FMK were obtained from BioVision (Mountain, PA, USA). The cell proliferation reagent WST-1 and RNase A were obtained from Roche Applied Sciences (Mannheim, Germany). Antibodies against cleaved caspase-3, phospho-ERK1/2, ERK1/2, phospho-JNK, and JNK were purchased from Cell Signaling (Beverly, MA, USA). Antibodies against phospho-p38 and p38 MAPK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The other chemicals were purchased from Sigma (St. Louis, MO, USA). The extraction and isolation of crude extract from Pinnigorgia sp. (Pin) and pinnigorgiol A were carried out as described previously [11 (link),16 (link)]. The ¹H and ¹³C NMR spectrum of pinnigorgiol A–C have been identified previously [16 (link)]. When Pin and pinnigorgiol A were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), the final concentration of DMSO in the cell experiments was 0.1% and did not affect the parameters measured.

To evaluate the effect of 11-dehydrosinulariolide on H1688, H146 and Beas2B cell growth, the cells were seeded at 1 × 10⁵ cells/well in a 24-well plate overnight and were treated with 11-dehydrosinulariolide at different concentrations (0, 5, 10, 25, and 50 μM) for 12, 24 or 48 h in the absence or presence of 20 μM of an irreversible caspase 3 inhibitor (zDEVD-fmk) (BioVision, Inc., Mountain View, CA, USA). After incubation, 200 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT; 0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and was incubated at 37 °C for 4 h. The purple formazan crystals produced by the mitochondrial dehydrogenase enzymes were dissolved in 600 μL of DMSO and were detected at 540 nm using an ELISA microplate reader (Tecan Sunrise, San Jose, CA, USA). The concentration of 11-dehydrosinulariolide required to inhibit cell proliferation by 50% (IC50) was calculated using Microsoft Excel software for semi-log curve fitting with regression analysis.