Apo one caspase 3 7 assay

For the caspase-3/-7 activity assay, CC cells (10,000 /well) were seeded on 96-well plates. After 48-h starvation, cell numbers and caspase-3/-7 activity in the same sample were monitored using CellTiter-Blue (Promega, Madison, WI, USA, #G8081) and Apo-ONE Caspase-3/7 assay (Promega, #G7790), respectively. Caspase-3/-7 activity was calculated as the ratio of Apo-ONE/CellTiter-Blue signals. The measurement was performed in triplicate.

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and cultured in endothelial cell medium containing supplements and 5% FBS (ECM; ScienceCell). Cells were cultured at 37 °C with 5% CO₂. HUVECs were transfected with control or PNUTS siRNA for 48 h (except for sprouting assays, see below). For proliferation assays, cells were incubated with 10 μM EdU for 4 h using the Click-iT EdU microplate Kit (Invitrogen) according to the manufacturer’s protocol. Apoptosis was assessed by incubating the cells with 200 nM Staurosporin or medium for 4 h and the caspase 3/7 activity was assayed using the ApoOne Caspase 3/7 Assay (Promega). PP1 was inhibited by stimulation with Tautomycetin (166 nM, R&D Systems). Senescence associated β-Galactosidase activity was analyzed with the Senescence Associated β-Galactosidase Staining Kit (Cell Signaling Technologies). Images were taken with a bright-field microscope (Axio Observer Z1.0 microscope, Zeiss) and the number of total cells, as well as the number of stained cells was determined in 4 images for per condition and experiment.

Cell proliferation was assessed using the MTS dye reduction assay (Promega Corp, Madison, WI). Briefly, 10 µl of MTS reagent was added to each well of the 96-well plate and incubated at 37°C for 60 min. The color change was assessed by measuring the absorbance value of each well at 450 nm with an ELx808 BioTek absorbance microplate reader (BioTek Instruments, Winooski, VT). The Apo-One Caspase-3/7 assay (Promega Corp, Madison, WI) was used to assess apoptosis as per the manufacturer's instructions.

D407 cells were plated at a density of 1.5×10⁴ cells/well in a 96-well plate. Cells were transfected 24 hrs after plating using Transfast reagent (Promega) with a 3∶1 (transfection reagent: DNA) ratio. Plasmid DNAs peGFP, or pTarget-BALB/CByJ Spink2 were used at a concentration of 100 ng/well. After 24 hrs, cells were treated with either 1 μM STS or DMSO for timepoints 0, 4, 8, 19 and 24 hours. STS treatments ended simultaneously, at which time, cells were incubated with Caspase 3/7 reagent from the APO-ONE Caspase 3/7 Assay (Promega) for 4 hours. Fluorescence was measured using a Tecan Safire² Microplate Reader (Tecan US, Inc, Durham, NC).