FIGURE 6.

Effects of proteinase K treatment on sEVs Wnt ligand contents. HT-22 cells-derived sEVs were treated with proteinase K in the absence and presence of Triton X-100 (see section “Materials and Methods”). (A) Proteinase K treatment did not affect the Wnt3a ligand presence. However, plasma membrane permeabilization with 0.5% Triton X-100 and co-treatment with proteinase K resulted in its disappearance. On the other hand, proteinase K treatment alone resulted in marked declines or even disappearance of Wnt5a, Wnt7a ligands, and the exosomal marker CD63. However, in the case of Alix, a band shift was produced (see comments in the section “Discussion”). Triton X-100 and co-treatment with proteinase K also resulted in the disappearance of CD63 and Alix. (B) The graphs show the relative protein expression levels with the different treatments. The staining intensity of each band derived from control P100 was assigned a value of 100%, and the value obtained in the P100 from the different treatments was compared with its respective control (100%). Wnt3a: 89.33 ± 8.02 (PK; p = 0.0638); 96.87 ± 5.32 (TX-100; p = 0.7695). Wnt5a: 0.41 ± 0.20 (PK; p = 0.002); 79.34 ± 30.33 (TX-100; p = 0.2904). Wnt7a: 3.02 ± 2.02 (PK; p < 0.0001); 85.14 ± 20.65 (TX-100; p = 0.2673). CD63: 7.23 ± 7.0 (PK; p < 0.0001); 85.73 ± 7.63 (TX-100; p = 0.0842). Alix: 40.72 ± 14.59 (PK; p < 0.0025); 86.23 ± 24.73 (TX-100; p = 0.5365). n.d., not detected; PK, proteinase K; TX-100, triton X-100. Mann–Whitney non-parametric t-student test (ns, not significant; ***p < 0.005; ****p < 0.0001). The images are representative of three independent cell culture preparations.