PMC

(A) Summarized data showing that pretreatment with TRAIL significantly attenuated acetylcholine (Ach)-induced endothelium-dependent relaxation in small arteries isolated from Smpd1^+/+ mice. (B) Summarized data showing that pretreatment with TRAIL had no inhibitory effect on acetylcholine (Ach)-induced endothelium-dependent relaxation in small arteries isolated from Smpd1^−/− mice. *P<0.05 vs. Smpd1^+/+ control (n=6 mice).

(A) Representative confocal images depicting the effect of TRAIL on the co-localization between MRs (detected by CTXB) and ASM in Smpd1^+/+ and Smpd1^−/− CAECs. (B) Displaying are the summarized data showing the colocalization between MRs and ASM. (C) Representative confocal images depicting the effect of TRAIL on the co-localization between MRs and ceramide in Smpd1^+/+ and Smpd1^−/− CAECs. (D) Displaying are the summarized data showing the colocalization between MRs and ceramide from six independent experiments. *P < 0.05 vs. Smpd1^+/+ control; #P < 0.05 vs. Smpd1^+/+ TRAIL (n=6).

(A) Representative confocal fluorescence images showing the colocalization between MRs and gp91^phox in Smpd1^+/+ and Smpd1^−/− CAECs. (B) Displaying are the summarized data showing the colocalization between MRs and gp91^phox from six independent experiments. *P < 0.05 vs. Smpd1^+/+ control; #P 0.05 vs. Smpd1^+/+ TRAIL (n=6).

CAECs were stimulated with or without TRAIL and then incubated with O₂^−· -specific spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) to form more stable free radicals (CMH-O₂^−·), which was immediately analyzed by electron spin resonance spectrometry (ESR). Summarized data showing the effect of TRAIL on O₂^−· production in Smpd1^+/+ and Smpd1^−/− CAECs., *P < 0.05 vs. Smpd1^+/+ control; #P < 0.05 vs. Smpd1^+/+ TRAIL (n=6).

(A) Representative confocal images showing TRAIL induced clustering of MR markers ganglioside GM1, which were detected by cholera toxin subunit B (CTXB). (B) Summarized data showing the dose-dependent effect of TRAIL (0–100 ng/ml) on MR clustering in Smpd1^+/+ CAECs (n=6). (C) Confocal microscopy of Smpd1^+/+ CAECs revealed a co-localization of DR4 but not DR5 with MR clusters upon TRAIL stimulation. (D) Summarized data showing the co-localization between MRs and DR4 or DR5 in mouse Smpd1^+/+ CAECs. *P < 0.05 vs. control (n=6).

(A–C) Representative confocal images of fluorescence resonance energy transfer (FRET) between FITC-Lamp-1 and TRITC-CTXB in Smpd1^+/+ CAECs treated with vehicle control (PBS buffer only) (A), TRAIL (B), or in Smpd1^−/− CAECs with TRAIL. The FRET images were obtained by subtraction of the pre-bleaching images from the post-bleaching images and shown in dark blue color. Increased intensity of blue color represents higher level of FRET in these cells. (D) Displaying are the summarized data showing the effect of TRAIL on FRET efficiency between FITC-Lamp-1 and TRITC-CTXB in Smpd1^+/+ and Smpd1^−/− CAECs. *P<0.05 vs. Smpd1^+/+ control; #P < 0.05 vs. Smpd1^+/+ TRAIL (n=6).

CAECs were first loaded with FM1–43, a lysosome specific fluorescence probe, and then incubated in fresh medium containing bromide phenol blue (BPB), which reversibly quench FM1–43 fluorescence. In quenching experiments, lysosome fusion with the plasma membrane will allow BPB to enter the lysosomes to quench FM1–43 fluorescence. (A) Left panel: representative confocal fluorescence images of FM1–43 fluorescence in Smpd1^+/+ CAECs before and after treatment with TRAIL. Right panel: representative traces of TRAIL-induced changes of FM1–43 fluorescence, normalized to that obtained before TRAIL treatment. (B) Left panel: representative confocal fluorescence images of FM1–43 fluorescence quenching in Smpd1^−/− CAECs treated with TRAIL. Right panel: representative traces of TRAIL-induced changes of FM1–43 fluorescence in Smpd1^−/− CAECs. (C) Summarized data showing the effect of TRAIL on FM1–43 fluorescence quenching in Smpd1^+/+ and Smpd1^−/− CAECs. *P<0.05 vs. Smpd1^+/+ control (n=6); #P < 0.05 vs. Smpd1^+/+ TRAIL (n=6).