Fig 1.
Structure of PSP1 peptide and binding of FITC-labeled PSP1 and annexin V to apoptotic cells.
(A) PEP-FOLD simulations give the structure of the PSP1 peptide (magenta; hydroxyl group: red/white). PS (white) is displayed with its head group (phosphate: yellow; amide: blue) toward PSP1. (B) PS exposure on the cell membrane of apoptotic cells is examined by FACS analysis based on shifting following binding to annexin V-Alexa Fluor 488 (X-axis) and PI (Y-axis). (C) The indicated amount of FITC-CLSYYPSYC or FITC-annexin V was incubated with apoptotic H460 cells (red) or normal H460 cells (black) at 4°C for 1 hr. Control peptide (grey); FITC-NSSSVDK. (D) 10 μg/ml of FITC-PSP1 peptide (CLSYYPSYC) and mutants FITC-PSP1 (AA-PSP1; CLSAAPSYC. FF-PSP1; CLSFFPSYC) were incubated with apoptotic H460 cells (red) or normal H460 cells (black). (E) Bar chart indicates the FITC labeled peptide binding (under bar in D) to apoptotic H460 (red) vs. normal H460 cells (black). The results are presented as the means + s.d. (n = 3 independent examinations per group) **P<0.01.
Fig 2.
In vitro SPR analysis of PSP1 and annexin V binding to PS liposomes.
(A) The indicated concentrations of PSP1 were injected onto the liposome-immobilized surface. Responses (RU) from the PC liposome-coated surface (reference) were subtracted from those from the PS:PC (2:8) liposome surface to monitor PS-specific binding. The equilibration binding constant (KD) and the association (Kon) and dissociation (Koff) rate constants were estimated using the Scrubber program. (B) Plots of the RU values of annexin V at the indicated concentrations.
Fig 3.
Microscopic analysis of binding of FITC-labeled PSP1 and annexin V to apoptotic cells.
(A) Apoptotic H460 cells in a chamber slide were incubated with 20 μg of PSP1, control peptide, or annexin V for 30 min. The cells were washed and fixed with 4% PFA before staining with DAPI. The confocal images were taken by laser confocal microscopy. (B) Normal H460 cells were subjected to incubation with the same amount of peptide or annexin V.
Fig 4.
In vivo imaging of tumors after apoptosis induction.
(A) Cy7.5-labelled PSP1peptide, control peptide, or annexin V was systematically injected into the tail vein of tumor-bearing nude mice treated with camptothecin 24 hrs before injection. The same amount of annexin V was also injected into non-camptothecin-treated tumor-bearing mice as a control. The homing of the peptides was examined after the indicated times using an optical imaging system. The white dotted circles indicate the tumors. (B) Histological examination of tumor tissues after homing of PSP1 and annexin V, performed under a fluorescence microscope. Tumor cell apoptosis was observed by TUNEL staining.