Z lehd fmk

EC109 cells were seeded at 4×10⁵ cells/well in 6-well plates and incubated for 24 h until adherent. Next, the cells were treated with JD or Oridonin at 15 μM and 30 μM, with control, which was left untreated for 24 h. After drug treatment, adherent and non-adherent cells were harvested, and the Annexin V-FITC Apoptosis Detection Kit (Biovision, San Francisco, USA) was used according to the manufacturer’s protocol. Briefly, cells were washed with ice-cold PBS, resuspended in binding buffer containing Annexin V-FITC and propidium iodide, and incubated for 30 min at 37°C in the dark prior to analysis using a flow cytometer. Cells that were Annexin V-positive only and both Annexin V- and PI-positive were considered early and late apoptosis cells, respectively. Both the early and late apoptotic cells were included in the total number of apoptotic cells. All experiments were performed in triplicate.
For inhibition of Caspase-8 and Caspase-9, we used the Caspase-8 inhibitor, Z-IETD-FMK (Biovision, San Francisco, USA) and Caspase-9 inhibitor, Z-LEHD-FMK (Biovision, San Francisco, USA). EC109 cells were treated with Z-IETD-FMK or Z-LEHD-FMK 2 h before the addition of JD for 24 h. After drug treatment, adherent and non-adherent cells were harvested, stained with the Annexin V-FITC Apoptosis Detection Kit, and detected using flow cytometry. All experiments were performed in triplicate.

Tunicamycin and thapsigargin were purchased from Sigma and resuspended in DMSO. Tunicamycin was used at 0.1 µg/ml, and thapsigargin was used 0.1 µM, according to previous studies [15] (link), [37] (link). CD95 and camptothecin were purchased from BD Pharmingen and used at 5 µM per manufacturer's protocol. Caspase Inhibitor VI (Z-VAD-FMK) that inhibits all caspases was purchased from Calbiochem. Caspase-8/FLICE inhibitor (Z-IETD-FMK), Caspase-9/Mch6 inhibitor (Z-LEHD-FMK) and Caspase-12 inhibitor (Z-ATAD-FMK) were purchased from Biovision. All caspase inhibitors were dissolved in DMSO and kept at 2 mM as 1000X stock solution. For cell treatments, the aforementioned chemicals were diluted directly in corresponding media and filtered for sterile conditions.

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.

Heparinized human blood samples (350 μL) were pre-incubated with one of the following compounds for 1 hour: IM-54 (Enzo Life Sciences), Wortmanin, Genistein, Tyrphostin, PD098059, Necrostatin-5, Z-VAD-FMK, AZD7762, Catalase, Tiron, Acetovanillone (Sigma), BAPTA/AM, YVAD-CHO, NS3694, Thapsigargin (Calbiochem), Z-IETD-FMK, Z-WEHD-FMK, Z-LEVD-FMK, Z-LEHD-FMK (BioVision), Z-YVAD-FMK (Santa Cruz). Concentrations were utilized according to previous reports and standardized to our conditions for optimal inhibitory performance. After treatment with the inhibitory compounds, samples were incubated with Br-LPS (1.3 pmol/mL) for 2 hours. Samples were further processed and analyzed by cytometry for cell death with Annexin V as described above.

Human melanoma cell line A7 (45 (link)) and human embryonic kidney cell line 293T (46 (link)) were cultured in high-glucose Dulbecco’s modified Eagle’s (DME) medium (11965084, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (10437028, Thermo Fisher Scientific). All transfection experiments were conducted using Lipofectamine 2000 transfection reagent (11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions. Typically, A7 cells (1.5 × 10⁵ cells/well) and 293T cells (8 × 10⁵ cells/well) were seeded in 6-well culture plates a day before transfection and were transfected with 1 μg of plasmid DNA (mixed with 2.5 μl of Lipofectamine 2000) and 3 μg of plasmid DNA (mixed with 7.5 μl of Lipofectamine 2000), respectively. The concentrations of the caspase-9 inhibitor Z-LEHD-FMK (1149-5, BioVision) and the caspase-8 inhibitor Z-IETD-FMK (1148-5, BioVision) used in the study were 100 μM or 200 μM.