Human apoptosis proteome profiler array

Proteome profiling was conducted using a Human Apoptosis Proteome Profiler™ array (R&D Systems, Minneapolis, MN, USA), consisting of a nitrocellulose membrane with duplicate spots of 35 apoptosis-related proteins. T24 and UMUC3 cells were treated with or without azacitidine (10 μM) for 24 h, lysed, and 400 μg total protein was used for each array following the manufacturer’s protocol. The membranes were incubated with horseradish-peroxidase-conjugated antibody, followed by a chemiluminescent detection reagent, and detected with the GE Healthcare ImageQuant LAS4000 instrument (Cytiva, Marlborough, MA, USA). Each experiment was repeated in duplicate, and the integrated density of the spots was quantitated using Image J software (National Institutes of Health, Bethesda, MD, USA).

The Human Apoptosis Proteome Profiler™ array (R&D Systems, Minneapolis, MN, USA), which is composed of a nitrocellulose membrane duplicate spot of 35 apoptosis-related proteins, was used in the present study. Total protein from NCCIT and NTERA2 cells treated with NPS-1034 0 and 40 μM for 24 h were extracted, and 400 μg of total protein were used for each array, per the manufacturer’s protocol. Each experiment was performed in duplicate, and the integrated density of the spots was quantitated by Image J software (National Institutes of Health, Bethesda, MD, USA).

The Proteome Profiler Human Apoptosis Array (R&D Systems) kit simultaneously detects the relative expression level of 35 apoptosis-related proteins. In short, the SNB-19 cells were treated with compounds 5 and 9 at a concentration of 0.5 µg/ml. All immunodetection steps were performed in accordance with the manufacturer’s instruction. The blots were detected using an enhanced chemiluminescence system using LI-COR C-Digit Blot Scanner.

The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [54 (link)]. The selected cell lines (GAMG and U373) were treated with IngC for 6 and 24 h, for stress array and 24 and 72 h for apoptosis array while using a concentration equivalent to 3 x IC₅₀ value of each glioma cell line. In addition, THE expression of some of the proteins was validated by western-blot analysis as described below.

8505C cells stably transfected with the TUSC2 or control plasmids were treated with 2.5 µM of staurosporine (Sigma-Aldrich) for 24 h, after which they were resuspended in 100 µl lysis buffer (R&D Systems, Minneapolis, MN) and quantified using the Bradford method. Cell lysates (400 µg) were added to the array membranes “Proteome Profiler Human Apoptosis Array”, obtained from R&D Systems, that had been incubated for 1 h with a blocking buffer, following the manufacturer’s instructions. The lysates were incubated with the blocked membranes overnight at 4 °C, and then each array was subjected to several washes for 10 min. Then, array membranes were incubated with detection antibody cocktail and streptavidin-HRP buffer followed by three washes. Plots were detected with Chemi Reagent Mix (R&D Systems) and images were obtained after autoradiographic exposure. Each apoptosis-related protein and control antibodies were spotted on the membrane in duplicate (R&D Systems). Quantification of the array was performed with the ImageJ software programme (version 1.50i) and each protein is represented as the average signal (pixel density) of the pair of duplicate spots. Pixel density was normalized by subtracting the average background signals.