PMC

Cells were treated with (A) 0–300 ng/ml TRAIL for 2.5 hours or (B) 0–50 µg/ml anti-DR5 antibody for 20 h. The resultant cells were analyzed by western blotting using antibodies specific to caspase 3 (C3), caspase 8 (C8), and CFP/YFP.

(A) MB231_CFP-YFP cells were treated with 0–100 µg/ml staurosporine for 20 h and 0–1.7 µg/ml camptothecin for 72 h. Dose-dependent changes in FRET signals were determined as in Fig. 4. (B) MTT assay was used to evaluate cell viability in response to treatment of parental_MB231 (filled black) and MB231_CFP-YFP (open clear) cells with 0–25 µg/ml staurosporine for 20 h and 0–7.7 µg/ml camptothecin for 72 h, respectively. (C) MB231_CFP-YFP cells were treated with chemotherapy agents (100 µg/ml) for 72 h. FRET measurement and analysis was performed as in Fig. 4. Statistical analysis was performed as in Fig. 1 and 2.

(A) Parental MB231 (filled black) and MB231_CFP-YFP (open clear) cells were treated with TRAIL (0–100 ng/ml) for 2.5 h or anti-DR5 antibody (0–200 µg/ml) for 20 h, at the indicated concentrations. Cell viability was determined by MTT assay. (B) Cells were treated as in A and analyzed for apoptosis by flow cytometry after staining with Annexin-V-APC and propidium iodide (PI). p-values were determined using a Student’s t-test. EC₅₀ values were determined using nonlinear curve fitting as described in the Materials and Methods section. For each EC₅₀ value, 95% confidence intervals reflecting the statistical accuracy are reported in .

MB231_CFP-YFP cells were treated with (A) 0–300 ng/ml TRAIL for 2.5 h and (B) 0–100 µg/ml anti-DR5 antibody for 20 h. FRET was calculated and is represented using bar graphs on the left side of the panel. EC₅₀ values were determined by nonlinear curve fitting to normalized FRET values plotted against the log values of TRAIL and anti-DR5 antibody concentration in molar (right side of the panel). (C) Confocal microscopy images showing changes in FRET in MB231_CFP-YFP cells treated with 0–25 ng/ml TRAIL for 2.5 h. A pronounced shift from YFP acceptor emission (yellow) to CFP donor emission (cyan) can be observed for cells treated with increasing concentration of TRAIL, with an excitation setting = 458 nm. Statistical analysis was performed as in Fig. 1 and 2.

(A) Schematic view of FRET assay. The FRET probe, CFP-linker-YFP, is expressed as a fusion protein containing CFP and YFP linked via a peptide sequence of 16 amino acids: GSGDEVDGGIETDGSG. The linker region contains recognition sequences for caspase 3 (DEVD) and caspase 8 (IETD), which can be selectively cleaved by its respective enzymes upon induction of intrinsic and/or extrinsic apoptosis pathways. (B) Immunoblot analysis of CFP-linker-YFP FRET probe stably expressed in MB231 cells. (C) Cells expressing CFP- linker-YFP FRET probe produced a significant and stable FRET signal, cells were incubated in PBS buffer for 0–24 h. (D) The CFP- linker-YFP FRET probe was immunopurified from MB231_CFP-YFP cell lysate and visualized by SDS-PAGE and Coomassie Blue staining (top) or by immunoblot analysis (bottom). (E) Changes in FRET were monitored when 100 nM purified CFP- linker-YFP protein was incubated with 100 nM recombinant active caspase 3 (C3), caspase 8 (C8), or caspase 2 (C2) for 3 h. pvalues were determined using a Student’s t-test.