Z devd fmk

We cultured breast cancer MCF-7 cells in small dishes and after 24 h of incubation the cells were treated with varying concentrations (1, 3, 10 and 30 μM) of compound 7. The cells were then further incubated for 48 h and then collected. The harvested cells were centrifuged at 1000 rpm for 5 min, washed thoroughly with ice cold PBS and were then subjected to fixation in 75% ethanol over night at 4 °C. After fixation the ethanol was removed and cells (about 1 × 10⁶) were re-suspended in PBS having RNase (25 µg/mL), then the cells were further incubated for another half hour in propidium iodide (50 µg/mL) at 37 °C and subjected to flow cytometer analyses. Cell cycle distribution was analyzed by ModFitLT (Verity, Software House, Topsham, ME, USA).
To determine whether cell cycle distribution is dependent upon caspase activation and the influence of the nucleoside addition to apoptotic activity of compound 7, a caspase 3 inhibitor (50 μM, Z-DEVD-FMK, Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50 μM, Z-VAD-FMK, Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µM of each) (dNTP Mixture, Beyotime Biotechnology) were added to the media with compound 7 (30 µM) to incubation for 48 h and followed by flow cytometer analyses as described above.

Acrolein was purchased from Shandong Xiya Chemical Industry Co., Ltd. (Linyi, China). 3-Methyladenine (3-MA) (PI3K inhibitor) and Z-DEVD-FMK (caspase-3 inhibitor) were purchased from MedChem Express Co., Ltd. (Monmouth Junction, NJ, USA). Annexin V-FITC apoptosis detection kits were purchased from Beyotime Biotechnology (Nanjing, China). Mitochondrial membrane potential (JC-1) assay kit was obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). The Cell Counting Kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. (Shanghai, China).

The Annexin V-FITC/PI apoptosis detection kit (Keygen Biotechnology, Nanjing, China) was used to detect the effects of compound 7. MCF-7 cells (3 × 10⁵ cells/well) were seeded in 6-well plates and cultured for 24 h. After treatment of cells with compound 7 (0, 3, 10, 30 μM) for 48 h, adherent and floating cells were collected and washed with ice cold PBS, then cells were centrifuged. The pellet was suspended with binding buffer (500 μL), stained with Annexin V-FITC (5 μL) and PI (5 μL), and then incubated for 15 min at room temperature in the dark. After incubation, cells were analyzed with a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) to determine the percentage of apoptotic cells.
To determine whether cell apoptosis is dependent upon caspase activation and the influence of the nucleoside addition on apoptotic activity of compound 7, a caspase 3 inhibitor (50 μM Z-DEVD-FMK, Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50 μM Z-VAD-FMK, Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µM of each) (dNTP Mixture, Beyotime Biotechnology) were added to the media with compound 7 (30 µM) and incubated for 48 h.

PFOS (C8F17KO3S, MW: 538.22, purity ≥ 98%, CAS: 2795-39-3, CAT#: 77282) was procured from Sigma-Aldrich (St. Louis, MO, USA). The caspase-3 inhibitor zDEVD-FMK (CAS: 210344-95-9, CAT#: HY-12466) and PERK inhibitor GSK2606414 (CAS: 1337531-36-8, CAT#: HY-18072) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).