Camptothecin

To examine the effect of the ncRNAs on apoptosis, MCF‐7 cells were additionally treated with the topoisomerase I inhibitor camptothecin (Biovision, Milpitas, CA, USA). Twenty‐four hours after transfection, MCF‐7 cells on a six‐well plate were treated with 4.8 μm camptothecin for 4 h and incubated for further 24 h to induce apoptosis. Nontransfected and transfected cells untreated or treated with camptothecin were incubated with 3 μL Annexin‐V‐FITC (BD Biosciences, San Jose, CA, USA) and 5 μL propidium iodide (Sigma‐Aldrich) in the dark at 4 °C for 15 min. Following incubation, 400 μL Annexin‐FITC binding buffer (0.1 m Hepes pH 7.4, 1.4 m NaCl, 25 mm CaCl₂ in PBS) was added to the cells. The cells were then analyzed on a FACSCanto II flow cytometer (BD Biosciences).

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.

Transfected (24 hour transfection) MCF-7 cells (on a 6-well plate) were treated with 6 μM of the topoisomerase I inhibitor camptothecin (Biovision, Milpitas, USA) for 4 hours. Twenty-four hours after induction of the camptothecin-mediated apoptosis, the cells were stained for flow cytometry using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, California, USA) according to the manufacturer`s instructions. The cells were then analyzed on a FACS CantoII flow cytometer (BD Biosciences).