Effect of oleandrin on NF-AT nuclear translocation, DNA binding, NF-AT-dependent luciferase activity, and FasL expression. U-937 cells were treated with 100-ng/ml oleandrin for different times. NF-AT was detected from cytoplasmic and nuclear extracts by Western blot and blots were re-probed for tubulin and CRM1 respectively (A). U-937 cells, treated with oleandrin for 12 h were incubated with anti-NF-AT antibody conjugated with FITC and DAPI and detected in Fluorescence microscope (B). U-937 and Jurkat cells were treated with oleandrin for different times and nuclear extracts were assayed for NF-AT DNA binding by EMSA (C). U-937 cells were transfected with Qiagen SuperFect reagent for 3 h with plasmids for NF-AT or FasL promoter DNA that had been linked to luciferase (NF-AT-Luciferase or FasL-Luciferase) and GFP. After washing, cells were cultured for 12 h. The GFP positive cells were counted and transfection efficiency was calculated. Cells, treated with oleandrin (100 ng/ml) for different times were extracted and assayed for luciferase as per Promega protocol (D & E1). FasL level was detected by RT-PCR followed by PCR using FasL and actin specific primers (E2) and Western blot (E3) from oleandrin-treated cells extract and culture supernatant. The culture supernatant blot was re-probed with anti-proteinase antibody

Effect of oleandrin on caspases activity and cell death. U-937 cells were treated with 100-ng/ml oleandrin for different times and procaspase 3, caspase 3, procaspase 8, and caspase 8 were detected by Western blot using specific antibodies (A). Caspase 3, 8, and 9 were detected from whole cell extracts using colorimetric substrate. The absorbance was taken at 405 nm and results represented in fold activation of caspases (B1). The amount of cytochrome c was detected from mitochondrial extract and cytoplasm from 100 ng/ml oleandrin-treated cells for different times by Western blot (B2). Nuclear fragmentation was detected from oleandrin (100 ng/ml for different times) treated U-937 cells by propidium iodide staining (C)

Effect of oleandrin on Na⁺/K⁺-ATPase activity and microvis-cosity. U-937 cells were treated with oleandrin and ouabain (100 ng/ml each) for 12 h; Na⁺/K⁺-ATPase activity was assayed from whole cell extract (A). U-937 cells, treated with oleandrin for 12 h were incubated with DPH for 2 h with moderate shaking. DPH binding was detected from cells in spectrofluorimeter (B). U-937 cells were treated with different lipid molecules (100 ng/ml) or oleandrin and lipid molecules mixture (100 ng oleandrin and 100 ng lipid molecule incubated for 4 h) for 72 h. Cell viability was detected by MTT assay and results represented as inhibition of cell viability in percentage (C)

Effect of oleandrin on calcineurin activity. U-937 cells were treated with 100-ng/ml oleandrin for different times and calcineurin activity assayed from cell extracts. Cells extract from oleandrin-treated (36 h) cells was incubated with cyclosporin A (2.5 μM) and calcineurin activity assayed

Effect of cyclosporin A and BAPTA-AM on oleandrin-mediated cell viability, Ca²⁺ release, and calcineurin activity. U-937 cells, incubated with cyclosporin A or BAPTA-AM (2.5 μM each) for 4 h and then treated with oleandrin (100 ng/ml) for 24 h in triplicate. Cell death detected by annexin V-PE and analyzed in FACS (A). Intracellular free Ca²⁺ detected using Fura-2AM as fluorescent probe in Fluorimeter (B). Calcineurin activity assayed from whole cell extracts (C)

Effect of Cyclosporin A, actinomycin D, z-VAD-fmk, ALLN, or anti-FasL antibody on oleandrin-mediated cell death. U-937 cells were treated with cyclosporin A (2.5 μM), actinomycin D (30 nM), z-VAD-fmk (3 μM), ALLN (10 μM), anti-FasL antibody (1 μg/ml) for 6 h and treated with 100 ng/ml oleandrin for different times. Cell death was assayed by Annexin V-PE and detected in FACS and represented in percentage. The figure represented one of the two independent experiments.

Effect of oleandrin on cell death, translocation of NF-AT to nucleus, and NF-AT DNA binding activity in Bcl-xL overexpressed cells. HL-60 (neo) and HL-60 (Bcl-xL) cells were treated with oleandrin (100 ng/ml) for different times. The level of Bcl-xL was detected by Western blot (A). Same blot was re-probed with anti-tubulin antibody. Cells were treated with oleandrin fro 12 h and NF-AT was detected by immunofluorecence assay (B). Cells were treated with oleandrin for different times and nuclear extracts were assayed for NF-AT DNA binding by gel shift assay (C). Cell viability was assayed by MTT uptake and indicated as inhibition of cell death in percentage (D)