Apoptotic Evaluations

Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling (TUNEL) staining. To measure Caspase-3 and -7 activities, MIO-M1 cells were seeded in a 96-well plate at a density of 1.5 × 10⁴ cells per well and incubated with 100 µL of conditioned media for 9 hours before siRNA transfection. After overnight incubation with siRNA, media were changed into serum-reduced ones (1% FBS) and incubated for 48 hours, followed by 10 ng/mL IL-1β application. Twenty-four hours later, Caspase-Glo 3/7 assay system reagent (Promega) was added to each well and luminescence rates were measured using a microplate reader (Tecan) according to the manufacturer's instructions.

For TUNEL staining, cells were seeded on 4-well culture slides at a density of 1.2 × 10⁵ cells per well. Following incubation in the same way as for the Caspase-3/7 assay, cells were fixed in 1% paraformaldehyde and stained for TUNEL (ApopTag fluorescein in situ apoptosis detection kit; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Lonza, Basel, Switzerland). Images were captured under a Biorevo microscope (BZ-9000; Keyence, Osaka, Japan). The number of total nuclei and TUNEL-positive cells were counted using ImageJ software (NIH) in the two views of each well. The rates of TUNEL-positive cells in total nuclei were averaged per well before statistical analysis.