Apo one homogeneous caspase 3 7 assay kit

Apoptotic cell death was measured with an Apo‐ONE Homogeneous Caspase‐3/7 assay kit (Promega) according to the manufacturer's guide. The absorbance was detected at 520 nm using a SpectraMax i3x microplate reader (Molecular Devices). The experiments are repeated three time independently.

Activities of caspase-3/7 and caspase-9 were assessed using an Apo-ONE™ homogeneous caspase-3/7 assay kit (Promega, Madison, WI, USA) and a Caspase-Glo™ 9 assay kit (Promega), respectively, according to the manufacturer’s protocols [16 (link), 17 (link)]. Briefly, cells were seeded into 96-well plates (2 × 10⁴ cells/well) and incubated overnight. On the following day, the cells were treated with the indicated concentrations of the appropriate drug for varying times. After the treatment, 100 μL of the Apo-ONE™ caspase-3/7 reagent was added to each well. The plates were incubated at room temperature for 1 h, and the luminescence of each well was measured using an EnSpire™ multimode plate reader (PerkinElmer, Waltham, MA, USA). Caspase activity was calculated by comparing the levels of luminescence of the treated cells with that of the control cell population incubated without drugs, which was defined as 100%.

For the first assay, the Apo-one Homogeneous Caspase-3/7 Assay kit (Promega Corporation, Madison, WI, USA) was used following the manufacturer’s instructions in order to estimate caspase 3/7 activity in PDAC cells (MiaPaCa-2 and Panc-1) after CDF and CDFHCD treatment. In the second assay, Annexin V/PI staining was performed and studied using flow cytometry. Briefly, 1 × 10⁵ PDAC cells were plated in 6-well plates. After 24 h of plating, PDAC cells (MiaPaCa-2 and Panc-1) were treated with vehicle control, CDF, and CDFHCD at IC₅₀ doses. At the end of the treatment, cells were washed and stained with Annexin V-FITC antibody and propidium iodide (PI), as per the manufacturer’s protocol, and were studied by flow cytometry.

Caspase activity/apoptotic activity was determined using the Apo-One homogeneous caspase-3/7- Assay kit (Promega, #G7790): 10.000 cells per well were seeded, grown for 24 h and incubated for 24 h with different concentrations of BYL719. Concentrations of 10 μM (effective dose close to the IC50 of the cell viability experiments) and higher doses were tested. Caspase-3/7 activity was assessed following the manufacturer’s instructions. For statistical analysis, a priori tests evaluating the normal distribution and homogeneity of variances were performed by the Kolmogorov-Smirnov-Test and the Levene′s Test of the SPSS statistical package SPSS (version 13.0 for Windows, SPSS Inc (2005), Chicago, USA). Non-parametric criteria were met; therefore, the Kruskal-Wallis followed by the Mann-Whitney test was performed. Statistical significance was assessed at p<0.05. Three independent samples per data point have been analyzed; each experiment was repeated at least two times in an independent manner. The results are displayed as mean ± SD.

The activities of caspase-3 and caspase-7 were determined using an Apo-ONE Homogeneous Caspase-3/7 Assay Kit (Promega), according to the manufacturer’s protocols (Okamoto et al., 2016 (link); Sumi et al., 2018b (link)). In brief, cells were seeded at 2 × 10⁴ cells/well on 96-well plates and incubated overnight. Cells were then treated with the experimental concentrations of camptothecin and propofol for varying lengths of time. Caspase activities were determined by comparing the luminescence values of the treated cells with those of the control cells (incubated without drugs), with the latter defined as 100%. All assays were conducted in triplicate and repeated at least twice. Detailed protocols are available as Supplemental Information and at protocols.io (10.17504/protocols.io.v7je9kn).