FIGURE 4.

Distinct heparinase I and III sensitivity to CTX A2/A4 retention and endocytosis indicating the involvement of different HS domains. A, retentions of different CTX on immobilized cell surfaces were dependent on surface GAG and were sensitive to different heparinase treatments as revealed by the reduced length of the blots (three individual repeats to show retained CTX). B, endocytotic CTX A2 and CTX A4 inside the H9C2 cells was located at the lysosome (as indicated by LysoTracker) but not at the mitochondria (as indicated by MitoTracker). Bar = 20 μm. C, endocytosis of different CTX into H9C2 or CHO cells was dependent on surface GAG and was sensitive to different heparinase treatments. Bar = 5 μm. D, CTX A2 and A4 induced lysosomal membrane permeabilization. CTX A2 or A4 decreased the red fluorescence of acridine orange (upper panels; green fluorescence was from LysoTracker Green DND-26) and the green fluorescence of LysoSensor DND-189 (middle panels) as compared with naphthazarin (lysosomal destabilizer, 5 μm). Treatment with naphthazarin released cathepsin D into the cytosol, but this was not observed for CTX A2 orA4 (intact spots remained). Data were mean ± S.E. (n > 10); **, p < 0.01.