Caspase glo 9 assay system

The cells were subjected to measure the activity of caspase 8 and caspase 9 using a specific-substrates, luminescent-based commercial kit, Caspase-Glo^® 8 Assay and Caspase-Glo^® 9 Assay Systems from Promega. Briefly, the cells in 100 μl of culture medium were combined with 100 μl of Caspase-Glo^® 8 or Caspase-Glo^® 9 working reagents. The reagents provide lysis buffer, luminescent-conjugated substrates of caspase8 or caspase 9 and MG132. MG132 was added to lower the background signal. The reaction mix was incubated for 30 minutes at room temperature and total luminescent light was measured using Synergy H1 Multi-Mode Reader (BIOTEK, Winooski, VT, USA)

Solvents for the synthesis and purification were purchased from Sigma-Aldrich (Poznan, Poland). All cell culture reagents were purchased from Gibco^® (Life Technologies Polska Sp. z o. o., Warsaw, Poland). Flasks and multiwell transparent and black plates for in vitro studies were obtained from Nunc (Life Technologies Polska Sp. z o. o., Warsaw, Poland). PAMAM G4-NH₂ dendrimer, docetaxel, paclitaxel, phosphate buffered saline (PBS), fetal bovine serum (FBS), propidium iodide (PI) and ribonuclease A deoxyribonuclease-free were purchased from Sigma-Aldrich (Poznan, Poland). Trypan blue was purchased from Molecular Probes ((Thermo Scientific™, Warsaw, Poland). Annexin V and 2′,7′-dichlorodihydrofluorescein (H2DCF-DA) were purchased from BD Biosciences (Warsaw, Poland). Caspase-Glo^® 3/7 Assay, Caspase-Glo^® 8 Assay and Caspase-Glo^® 9 Assay systems were purchased from Promega Corporation (Mannheim, Germany). Trastuzumab (Herceptin) was obtained from Roche Poland (Poznan, Poland). Human breast adenocarcinoma′s cell lines, including HER-2 positive (SKBR-3 ATCC no. HTB-30) and HER-2 negative (MCF-7 ATCC no. HTB-22) were purchased from ATCC (LGC Standards Sp. z o. o., Lomianki, Poland).

Human ovarian carcinoma cell lines A2780/CP70 and human immortalized ovarian surface epithelial cells (IOSE 364), were kind gifts from Dr. Bing-Hua Jiang at Thomas Jefferson University and Dr. Auersperg at the University of British Columbia, respectively. Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO, USA) incorporating 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY, USA). Cells were grown in a humidified incubator containing 5% CO₂ at 37 °C.
Reagents: Kaempferol was purchased from Sigma. Dead Cell Apoptosis Kit with Annexin V Alexa Fluor^® 488 and propidium iodide (PI) was purchased from ThermoFisher Scientific (Waltham, MA, USA). Caspase-Glo^® 3/7 Assay Systems, Caspase-Glo^® 8 Assay Systems and Caspase-Glo^® 9 Assay Systems were purchased from Promega (Madison, WI, USA). Antibodies against p21 Waf1/Cip1 (p21), p-Cdc25C (Ser 216), Cdc25C, p-Cdc2 (Tyr 15), Cdc2, Cyclin B1, p53, death receptor 5 (DR5), Fas, and Fas-Associated protein with Death Domain (FADD) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against phosphor-checkpoint kinase 2 (Chk2) (Thr68), Chk2, poly [ADP-ribose] polymerase 1 (PARP-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Cells were treated with a common cytokine mix containing 185 U/ml TNF-α, 60 U/ml IL-1β and 14 U/ml IFN-γ (all ReliaTech, Wolfenbüttel, Germany) [1 (link), 24 (link)] for 12 h, 24 h or 48 h in their respective differentiation or culture media. Apoptosis was assessed using the Caspase-Glo 3/7, the Caspase-Glo 8 or the Caspase-Glo 9 Assay Systems or the RealTime-Glo Annexin V Apoptosis Assay (all Promega, Walldorf, Germany) according to the manufacturer’s instructions. For details see the ESM Methods.
NO was measured as accumulated nitrite in the medium by the Griess reaction and intracellular reactive oxygen species (ROS) were estimated using the fluorescent probe dichlorofluorescein diacetate (DCFDA, Thermo Fisher Scientific), as described in detail in the ESM Methods. Definitive endoderm (DE) cells were derived from HES-3 and SC-organoids were derived from HES-3 SC30 ICNC4.

Caspases 3/7 and 9 activities were assessed using a fluorescence based Apo-ONE Homogeneous Caspase-3/7 Assay Kit (Promega) and luminescence-based Caspase-Glo 9 Assay System (Promega), respectively, following manufacturer’s protocol. Briefly, 2.5 × 10⁴ cells were seeded onto 96-well plates, allowed to adhere overnight, and treated according to indicated drug schedules. 120 μL of master reagent (mix of kit’s substrate 170 and buffer) was loaded onto each well, gently mixed in a shaker for 1 min, and incubated for 40 min to 90 min at RT. Excitation and emission wavelengths were set at 560 and 590 nm, respectively. Luminescence was read on POLARstar Omega luminometer.