Caspase 3

Following a 24 h of treatment of YUMM 1.7 cells (350000 cells per well in a 6-well plate) with 150 μg/mL of extract or 2 μM of etoposide, cells were lysed using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 2 mM EDTA). Protein content was determined using the BCA assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Samples (nontreated cells and cells treated with either extract or etoposide) were loaded and run in a polyacrylamide gel for 90 min, at 100 V. Separated proteins were then transferred to a PVDF membrane and blocked using Intercept Blocking Buffer (LI-COR). The membrane was then incubated with a primary antibody from rabbit (caspase-3, Cell Signaling Technology) and mouse antibody for β-actin (Cell Signaling Technology) for a period of 24 h in a cold room. The membrane was then washed 3 times with PBST (PBS + 0.1% Tween 20) and incubated with the secondary antibody; the donkey antirabbit antibody (cell signalling technology) was conjugated to a chemiluminescent such as IR680 (red) to detect cleaved caspase-3, and the donkey antimouse antibody conjugated to IR800 (green) to reveal β-actin. The plate protected from light was incubated for 1 h and later visualised using the Odyssey system associated with ImageStudio (LI-COR).

Homogenized total renal tissue or NRK-52E cells were lysed in a sodium dodecyl sulfate sample buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 1.5 mM MgCl2; 1 mM ethylene glycol tetraacetic acid; 10% glycerol; 1% Triton X-100; 1 μg/mL aprotinin; 1 μg/mL leupeptin; 1 mM phenylmethylsulfonyl fluoride; and 0.1 mM sodium orthovanadate) at 4°C [18 (link)]. Protein (50 μg/sample) was transferred to a nitrocellulose membrane and probed with the appropriate primary antibodies (anti-LC3 [MBL], anti-HSPB1 and phospho-specific anti-S82-HSPB1 [Cell Signaling Technology Inc.], anti-actin [Santa Cruz Biotechnology, Inc.; catalog #sc-10731], caspase 3 [Cell Signaling Technology Inc.; catalog #9665], a 1:1000 dilution of cleaved caspase 3 [Cell Signaling Technology Inc.; catalog #9664], activated Bax [6A7; Trevigen, Inc., Gaithersburg, MD; catalog #2281-MC-100], CHOP [Cell Signaling Technology Inc.; catalog #785324], or total Bax [5B7; Pharmingen; catalog #556467]). The primary antibodies were detected using horseradish peroxidase-linked anti-rabbit immunoglobulin G and visualized using an Amersham ECL system (Amersham Corp., Arlington Heights, IL). We performed densitometric analysis of LC3-II:actin and SQSTM1:actin.

The CT26 cell lysates from the variety of the treatments were prepared using RIPA lysis buffer for immunoblotting of Cyclin D1 (Cell Signaling Technology, #92G2), Cyclin A (Santa Cruz Biotechnology, #sc-271,645), Hsp70 (Santa Cruz Biotechnology, #SC24), Caspase-3, and cleavage form of Caspase-3 (Cell Signaling Technology, #9662S). Western blot analysis was performed as previously reported [38 ].