Figure 3.

In vitro generation of apoptosis-specific azido groups of Apo-S-Ac₃ManNAz-treated PC-3 tumor cells. (a) Time-dependent TRAIL-induced apoptosis was analyzed using western blot analysis of Cas-3, Cas-8 and GAPDH, wherein PC-3 tumor cells were incubated with TRAIL for 0, 1, 3, 6, 9, and 24 h for inducing apoptosis. (b) Time-dependent generation of azido groups was analyzed by western blot analysis of Apo-S-Ac₃ManNAz- and TRAIL-treated PC-3 tumor cells. (c) Time-dependent CLSM images of Apo-S-Ac₃ManNAz (20 μM) and TRAIL (7 ng/ml)-treated PC-3 tumor cells, followed by DBCO-Cy5.5 (200 nM) to visualize azido groups. Red = DBCO-Cy5.5 channel; Blue = DAPI channel. (d) The MFI of Apo-S-Ac₃ManNAz- and TRAIL-treated PC-3 tumor cells at various incubation time. (e) CLSM images of apoptosis-specific Apo-S-Ac₃ManNAz-treated PC-3 tumor cells after post-treatment of TRAIL or TRAIL with z-DEVD-FMK at 24 h. Red = DBCO-Cy5.5 channel; Blue = DAPI channel. (f) MFI of flow cytometry analysis of Apo-S-Ac₃ManNAz- and TRAIL-treated PC-3 tumor cells without/with z-DEVD-FMK at 24 h. (g) Western blot analysis of generation of azido groups of Apo-S-Ac₃ManNAz-treated PC-3 tumor cells after post-treatment of TRAIL or TRAIL with z-DEVD-FMK at 24 h.