Fig 1: Effect of recombinant human PPT1/TPP1 protein on reducing enlarged lysosomes in NCL patient NSCs. The western blot analysis (a, b) showed that there is a PPT1 deficiency in PPT1E8/E1 fibroblasts and NSCs, and also there is no TPP1 expression detected in TPP1E4/E6 and TPP1E4/IVS5 fibroblast and NSCs. The treatment of NCL NSCs with 200 nM rPPT1/rTPP1 significantly reduced the LysoTracker dye staining(c), with an effect nearly 99.9% in the NCL NSC lines treated with ERT (d). The images were taken with 40X objective lens. Data are displayed as mean ± SD. ** P < 0.01
Fig 2: 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
Fig 3: Effect of DT, HPBCD and enzyme replacement therapy on the accumulation of subunit c in patient NSCs. Cells were treated with 20 μM DT, 1 mM HPBCD, 20 μM DT plus 125 μM HPBCD, or 200 nM rPPT1/rTPP1 for 3 days. PPT1/TPP1 expression were restored after the PPT1/TPP1 replacement therapy. After the treatment with 20 μM DT plus 125 μM HPBCD, the expression of subunit c in PPT1E8/E1NSCs decreased by 75% (a and b). Moreover, it showed that subunit c expression in TPP1E4/E6 NSCs and TPP1E4/IVS5 NSCs was decreased by 51% and 64% respectively, in TPP1 replacement treatment, and also decreased by 18%, 30% with DT treatment (c, d, e and f). Data are displayed as mean ± SD. * P < 0.05, ** P < 0.01
Supplier Page from Sino Biological, Inc. for Human PPT1 / Palmitoyl-protein thioesterase 1 Protein (His Tag)