Screening and characterizing TNF peptides for the induction of apoptosis and necrosis. (A,B) Cell death induced by selected 20-mer TNF peptides (P12–P18). Each of the overlapping peptides was incubated overnight at 50 µM with Jurkat A3 (A) or L929 (B) cells. Apoptosis and necrosis were measured by using flow cytometry analysis of active caspase-3 and membrane integrity (live–dead cell staining), respectively. The experiments were repeated more than five times. (C) z-VAD inhibited peptide-induced apoptosis. Jurkat A3 cells were incubated overnight with 100 µM of P12, P13 and P18, in the presence of 10 µM z-VAD. The experiment was repeated three times. (D) Apoptosis induced by the 30-mer peptide P1213. Jurkat A3 cells were incubated with the 30-mer peptide P1213 at 30 µM overnight, with or without the presence of 10 µM z-VAD, P18 was used as a control peptide. The experiment was repeated three times. (E) DNA fragmentation coincided with active caspase-3 expression in P12- and P13-induced apoptosis. P12- and P13-induced apoptosis is shown in fluorescence images of DAPI-stained nuclei with DNA fragmentation (upper panel) and fluorescence-labeled active caspase-3 (lower panel). White arrows point to nuclear DNA fragmentation (upper panel), which coincides with caspase-3 activation in the cells without z-VAD (lower panel). Scale bar: 20 µm. The experiment was repeated twice. (F) Transmission electron microscopy analysis of TNF-peptide-treated cells. Jurkat A3 cells were treated with selected peptides (P12, P13, P15 and P16) at 100 µM. Cells cultured with P12 and P13 show DNA fragmentation, blebbing and membrane shedding, whereas cells treated with P15 and P16 show necrosis-like features, such as loss of cell membrane integrity.

Cell death induced by peptides is independent of TNFR1. (A) Confocal microscopy images showing that the peptides do not bind to TNFR1. Biotinylated P1213 or P16 were incubated with Jurkat A3 cells; biotinylated TNF was used as a positive control. After incubation with cells, peptides or TNF were stained with avidin conjugated with Alexa-Fluor-488. TNFR1 was stained with an antibody against TNFR1, followed by a second antibody conjugated with Alexa-Fluor-568. Scale bar: 10 μm. (B) P1213 does not cause TNFR1 to co-immunoprecipitate with TRADD. Jurkat A3 cells stimulated with P1213 or TNF at the indicated times were subjected to immunoprecipitation (IP) with an antibody against TRADD, followed by western blotting (WB) for TNFR1. (C) L929 cells were incubated with 20 ng/ml TNF or 50 µM P1213 in the presence or absence of an antibody against TNFR1 (Anti-TNFR1; 20 µg/ml) overnight. (D) Functionally, P1213-induced apoptosis is independent of siRNA-mediated TNFR1 (siRNA-TNFR1) inhibition. Jurkat A3 cells that had been treated with or without siRNA-TNFR1 were incubated with P1213, and active caspase-3 in the cells was then measured. The right-hand panel shows inhibition of TNFR1 expression after siRNA-TNFR1 treatment by western blotting. All experiments were repeated at least twice. Means±s.d. of triplicate experiments are shown. P-values were calculated using Student's t-test.

P1213 enters cells and stimulates apoptotic complex-II formation. (A) P1213 enters cells. HT29 cells were treated with P1213 for 2 h before fixation. The cells were incubated with an antibody against E-cadherin (to label the membrane) and further stained with a secondary antibody conjugated to Alexa-Fluor-568. The biotinylated P1213 was stained with avidin-conjugated Alexa-Fluor-488. The confocal images (left and middle panels), representing two different layers of cells at 3.84-μm and 6.40-μm sections scanning from top, show that P1213 (green) is located both on the cell membrane and within the intracellular compartment. The right panel is a schematic picture showing different layers scanned from top of the cells with the confocal microscope. The distance between each layer is 0.64 μm. (B) P1213 binds to TRADD. Jurkat A3 cells that had been stimulated with biotinylated P1213 were stained with avidin–Alexa-Fluor-488 and an antibody against TRADD, and were then subjected to confocal microscopy. Scale bar: 10 μm. (C) P1213 co-immunoprecipitates TRADD with RIP1 or with FADD. Jurkat A3 cells that had been stimulated with P1213 or TNF for the indicated times were subjected to immunoprecipitation (IP) with an antibody against TRADD, followed by western blotting (WB) for RIP1 or FADD. The experiment was repeated twice. (D) P1213 does not induce apoptosis in RIP1^−/−, caspase-8^−/− or FADD^−/− cell lines. Jurkat A3 and Jurkat-A3-based knockout cell lines RIP^−/−, I9.2 (caspase-8^−/−) and I2.1 (FADD^−/−) were incubated with P1213 overnight, followed by measurement of intracellular active caspase-3. The error bars represent mean±s.d. of three experiments. P-values were calculated using Student's ttest. No pep, no peptide.

Membrane-induced P16 structural changes, aggregation and membrane disruption. (A) Molar circular dichroism spectra of selected peptides at pH 7.4 (normalized for the number of peptide linkages). The circular dichroism spectra of P15, P16 and P18 are shown. (B) Circular dichroism spectra of P16 (50 µM) in aqueous buffer solution (thick dotted line), neutral POPC:POPE liposomes (thin dotted line) or negatively charged POPC:POPS liposomes (solid line). (C,D) Hydrodynamic radius (nm) of P16 measured using dynamic light scattering in (C) hydrophilic conditions or (D) hydrophobic conditions. (E) P16 disrupts cell membranes of RBCs. RBCs (10⁷/100 µl) were incubated with peptides at a concentration of 100 µM overnight. Lysis of RBCs by P16 or H₂O was observed, but not by P12, P18 or PBS. (F) P16 disrupts cell membranes of RBCs. RBCs (10⁷/100 µl) were incubated with different concentrations of peptides overnight. Protein (mainly hemoglobin) in the supernatant was measured to indicate cell lysis. The experiments were repeated three times. (G) P16 (50 µM) was incubated with negatively charged POPC:POPS liposomes or pH-neutral POPC:POPE liposomes encapsulating a fluorescent marker (Phen Greek SK). Disruption of the membrane was measured by the quenching of fluorescence intensity inside liposomes (fluorescence decreased, arrow). The error bars represent mean±s.d. of three experiments.