Article Figures & Data
Figures
- Figure 1.
Cell death in lung cancer cells and PBMCs induced by treatment with Apo2L/TRAIL and different concentrations of oleandrin. A, cell lines and PBMCs were treated for 48 hours and cell death was assessed by PI staining followed by flow cytometry. Points, mean of three independent experiments with duplicates; bars, SE. Two-way ANOVA was done to compare the effect of oleandrin concentrations and the effect of absence or presence of Apo2L/TRAIL treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, activation of caspases and cleavage of their substrates as shown by Western blot analysis. C, measurement of caspase-3 activity by determination of hydrolysis of DEVD-amc. Columns, mean of two independent experiments; bars, SE. ***, P < 0.001 (two-way ANOVA).
- Figure 2.
Apo2L/TRAIL receptor surface expression (A) and DR4 and DR5 mRNA expression (B) in Calu1 cells. A, cells were treated with 160 ng/mL oleandrin and 100 ng/mL Apo2L/TRAIL alone or in combination for 24 hours. Subsequently, cells were stained with monoclonal antibodies raised against the extracellular domain of Apo2L/TRAIL receptors DR4, DR5, DcR1, and DcR2. Data were analyzed by flow cytometry. Gray histogram, cells stained with isotype control IgG antibody. B, cells were treated with 160 ng/mL oleandrin and 100 ng/mL Apo2L/TRAIL alone or in combination for indicated time points and then harvested for extraction of total cellular RNA. DR4 and DR5 mRNA expression was detected by RT-PCR. Amplification of 28S rRNA mRNA was carried out as an internal control. Semiquantitative evaluated DR4 and DR5 mRNA levels of oleandrin-treated samples were expressed as fold increase versus their time-corresponding samples that were not treated with oleandrin. Representative of three independent experiments analyzed using two-way ANOVA.
- Figure 3.
Silencing of oleandrin-induced DR4 and DR5 mRNA expression (A) and DR4 and DR5 receptor expression on cell surface (B) and its effects on apoptosis induced by combined treatment with Apo2L/TRAIL and oleandrin (C). Transfected Calu1 cells were treated with 160 ng/mL oleandrin, and after 24 hours, the cells were either (A) harvested for preparation of total RNA and subsequent RT-PCR analysis or (B) stained with monoclonal antibodies and analyzed by flow cytometry. C, cell death was induced by combined treatment with 160 ng/mL oleandrin and 100 ng/mL Apo2L/TRAIL for 48 hours and assessed by PI staining followed by flow cytometry. Columns, mean of three independent experiments in duplicates; bars, SD. **, P < 0.01; ***, P < 0.001, significant difference to cells transfected with control siRNA.
- Figure 4.
Effect of inhibition of MAPKs [extracellular signal-regulated kinase 1/2 (ERK1/2), JNK, and p38] on expression of mRNA (A) and cell surface (B) expression of DR4 and DR5. Calu1 cells were preincubated for 30 minutes with 10 μmol/L U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 kinase inhibitor) and stimulated with 160 ng/mL oleandrin (Ole). After 24 hours, cells were either (A) harvested for preparation of total RNA extracts and subsequent RT-PCR analysis or (B) trypsinized and stained with monoclonal antibodies against DR4 or DR5 followed by flow cytometry. The effect of inhibitors of MAPKs on expression of mRNA expression of DR4 and DR5 receptors was semiquantitatively evaluated and expressed as fold increase relative to nontreated control cells. ***, P < 0.001, versus nontreated control cells; °, P < 0.05, versus oleandrin-treated cells; °°°, P < 0.001, versus oleandrin-treated cells. All experiments were done thrice.
- Figure 5.
Effect of different cardiac glycosides on Apo2L/TRAIL-induced apoptosis and expression of DR4 and DR5. A, Calu1 cells were treated with different concentration of cardiac glycosides alone or in combination with 100 ng/mL Apo2L/TRAIL for 48 hours. Cells were stained with PI for the assessment of cell death followed by flow cytometry analysis. Points, mean of three independent experiments with duplicates; bars, SE. Two-way ANOVA was done to compare the effect of different concentrations of cardiac glycosides and the effect of absence or presence of Apo2L/TRAIL treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Calu1 cells were treated with cardiac glycosides for 24 hours and stained with monoclonal antibodies raised against the extracellular domain of DR4 or DR5 followed by flow cytometry analysis.
Volume 66, Issue 11
