Fig. 7
Co-localization of subunit c and Lamp1 in NCL NSCs and the expression of subunit c in NCL NSCs. a Co-localization of LAMP-1, a lysosomal marker, with subunit c in PPT1E8/E1, TPP1E4/E6, and wild-type NSCs. The cells were immunostained with antibodies recognizing subunit c (red fluorescence, see white arrows) and Lamp1 (green fluorescence). Minimal overlap of subunit c and Lamp1 immunostaining was observed in wild-type cells (yellow in overlay), but Lamp1 strongly, though not perfectly, overlaps with the accumulated subunit c in PPT1E8/E1 and TPP1E4/E6 NSCs. Treatment of INCL and LINCL NSCs with recombinant PPT1 and TPP1 decreased subunit c accumulation in lysosomes of patient cells, respectively. Similar effects were also observed in cells after treatments with δ-tocopherol and HPBCD. Blue represents Hoechst nuclei stain. Images were captured with 60X objective. b and c Expression of subunit c in NCL fibroblasts analyzed by the Western blot. The expressions of subunit c in PPT1E8/E1 fibroblast were weaker than WT, but the expressions of subunit c increased in TPP1E4/E6 and TPP1E4/IVS5 fibroblast compared to WT (b). It showed that subunit c expression was decreased by 64% in PPT1E8/E1 fibroblast, and increased 1.5-fold in both TPP1E4/E6 and TPP1E4/IVS5 fibroblast compared to WT (c). Data are the mean ± SEM. ** P < 0.01. Expression of subunit c in NCL NSCs (D and E). The expressions of subunit c were weaker in PPT1E8/E1 NSCs than WT, but the expressions of subunit c were increased in TPP1E4/E6and TPP1E4/IVS5 NSCs compared to WT (d). It showed that subunit c expression was decreased by 36% in TPP1E4/E6 NSCs, and increased 1.2-fold in both TPP1E4/E6 and TPP1E4/IVS5 NSCs compared to WT (e). Data are displayed as mean ± SD. * P < 0.05, ** P < 0.01, compared to the WT control