Caspase 3 activity assay kit

Homocysteine, copper (II) chloride dehydrate, chloroquine (CQ), VAS2870, zVADfmk, propidium iodide (PI), Digoxin, Cytotoxicity detection kit and GAPDH (cat. no. G8795) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CM-H2DCF-DA, acridine orange, dihydroethidium, and Mitotracker red were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC) and caspase-3 activity assay kit were purchased from BD Biosciences (San Jose, CA, USA). Cytochrome c release assay kit was from EMD Biosciences-Calbiochem (San Diego, CA, USA). TransIT was from Mirus Bio (Madison, WI, USA). Atg7 siRNA, p62 siRNA, p22^phox siRNA and Lipofectamine RNAiMAX were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-cleaved caspase-3 (cat. no. 9664), anti-p62 (cat. no. 23214), anti-p22^phox (cat. no. 27297), and anti-Atg7 (cat. no. 8558) were from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3A (cat. no. NB 10-2331) was from NOVUS Biologicals (Littleton, CO, USA). Anti-PARP-1 (cat. no. 13371-1-AP) and anti-Beclin 1 (cat. no. 11306-1-AP) was from Protintech (Wuhan, China). Anti-p22^phox (cat. no. sc-271968) and anti-Bcl-2 (cat. no. sc-7382) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Caspase-3 activities were measured using a Caspase-3 activity assay kit (BD Biosciences, USA). The Caspase 3 assay kit is based on the hydrolysis of the substrate, acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety from the substrate, and released p-Nitroaniline (pNA) is detected at 405 nm. Comparison of the absorbance of pNA from the sample with the control allowed determination of the fold increase in caspase-3 activity and the relative caspase-3 activities are expressed in arbitrary units. SH-SY5Y cells encoding ATXN3-Q28 and ATXN3-Q84 were cultured in DMEM (Invitrogen, USA) containing 15% FBS and were cultured on 96 well dishes in DMEM (Invitrogen, USA) containing 15% FBS. Expression of ATXN3-Q28 and ATXN3-Q84 were induced in presence or absence of the drugs Ku55933 (2 μM), Pifithrin-α (10 μM) or STI-571 (5 μM) with doxycycline (500 ng/ml). The cells were harvested 3 days after induction and caspase-3 activities were measured. For measuring caspase-3 activities in the PNKP and control-siRNA-treated cells, the SH-SY5Y cells were cultured in 96-well dishes and transfected with PNKP-siRNA or control-siRNA in the presence or absence of Ku55933 (2 μM), Pifithrin-α (10 μM) or STI-571 (5 μM). The cells were harvested 48 hours after transfection, and caspase-3 activities were measured.