Human apoptosis array kit

The relative expression levels of 35 apoptosis-related proteins were evaluated using Human Apoptosis Array kit (R&D Systems, Abingdon, UK) in K562 cells. Proteins were extracted according to the manufacturer’s protocol from cells treated for 24 h with compound 15a (2 µM). The tool is fast, sensitive and economical, with 2.0 ml of array buffer added to each well, followed by array, The capture antibodies are retained in their specific locations, then incubated, transferred, diluted, added to Streptavidin-HRP, shaken. The platform shaker was incubated on the plate for 30 min, then the membrane was removed, 1 ml of the prepared Chemi Reagent Mix was evenly pipetted onto each membrane, incubated, the excess Chemi Reagent Mix was removed, and the membrane was placed in a self-developing film cartridge, exposed to X-ray film for 1–10 min. The positive signals seen on developed film can be quickly identified by placing the transparency overlay template on the array image and aligning it with the pairs of reference spots in three corners of each array. Creating templates, exporting files, averaging signals, finding backgrounds, comparing corresponding signals on the array to determine relative changes in apoptosis-related protein levels.

Analysis of phosphorylation profiles of kinases and their protein substrates, as well as analysis of expression of apoptosis-related proteins was done by the Human Phospho-Kinase Array (R&D Systems, Minneapolis, MN) and Human Apoptosis Array Kit (R&D Systems). For both, untreated and overnight 1 μg/ml cisplatin pretreated MSC were solubilized at 1 × 10⁷ cells/ml in lysis buffer at 2–8 °C for 30 min and proceeded according to manufacturer’s protocol. ImageJ software (NIH, Bethesda, MD) was used for the quantitative evaluation; pixel density was determined and calculated.
Cell supernatant of untreated MSC and pretreated MSC as above was analyzed by Human Cytokine Array Kit (R&D Systems) used to simultaneously detect the relative levels of 36 different cytokines, chemokines, and acute phase proteins according manufacturers protocol.

Cells were lysed in RIPA buffer after treatment with 2.5 μM 6-TG or vehicle. Equal amounts of protein were resolved by SDS-PAGE, then analyzed by western blotting with the indicated antibodies: anti-GAPDH (Proteintech, 60004-1-Ig), anti-DNMT1 (Genetex, GTX116011), anti-pAKT (abcam, ab38449), anti-AKT (abcam, ab8805) and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Proteintech, SA00006-4). The Human Apoptosis Array Kit (R&D, California, USA) was used for apoptosis array. The complete information about the antibodies is shown in Table S3.

To elaborate the underlying mechanism of apoptosis induced by Magnolol, a human apoptosis array was performed. HSC-3 cells (6 × 10⁵) were seeded in 6 cm dishes. After treatment with Magnolol (100 μM) for 24 h, cell lysates (400 µg) were collected using a Human Apoptosis Array Kit (ARY009) (R&D Systems, Minneapolis, MN, USA) according to the recommended protocol. The results detected the relative expression levels of 35 apoptosis-related proteins and provided 2 biological replicates normalized with internal controls.

Total cellular extracts were performed as previously described in Van Seuningen et al. [34 (link)] and Jonckheere et al. [35 (link)]. Western-blotting on nitrocellulose membrane (0.2 µm, Whatman, Maidstone, UK) was carried out as previously described [36 (link)]. Membranes were incubated with antibodies against STAT3 (79D7, Cell signalling), phospho S727 STAT3 (9134, signalling), c-Jun (60A8, Cell signalling), phospho S63 c-Jun (54B3, Cell signalling) and β-actin (AC-15, Sigma, Tokyo, Japan). Antibodies were diluted in 5% (w/v) non-fat dry milk in Tris-Buffered Saline Tween-20 (TBS-T). Peroxydase-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) were used and immunoreactive bands were visualised using the West Pico chemoluminescent substrate (Thermo Scientific, Pierce, Brebières, France). Intracellular signaling was studied using Human Phospho-Kinase Antibody Array (ARY003B, R & D) (detecting relative site-specific phosphorylation of 43 proteins) and Human Apoptosis Array Kit (ARY009, R & D). Chemo-luminescence was visualised using LAS4000 apparatus (Fujifilm, Tokyo, Japan). Density of bands were integrated using Gel analyst software^® (Claravision, Paris, France) and represented as histograms. Three independent experiments were performed.

Cell culture materials including Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Gaithersburg, MD, USA) and Hyclone Laboratories, Inc. (Logan, UT, USA), respectively. Antibodies specifically for p38, phosphorylated p38, caspase 3, propidium iodine (PI), and FITC (fluorescein isothiocyanate-labeled) Annexin V Apoptosis Detection Kit I were obtained from BD Biosciences (San Jose, CA, USA). The Human Apoptosis Array Kit was purchased from R&D Systems (Minneapolis, MN, USA). Additionally, antibodies specifically for ERK1/2, JNK1/2, phosphorylated ERK1/2 and JNK1/2, caspases 8 and 9, and cleaved caspases 3, 8, and 9 were purchased from Cell Signaling Technology (Danvers, MA, USA). Unless otherwise specified, all chemicals used in this study, including streptomycin, were purchased from Sigma-Aldrich (St. Louis, MO, USA).