r ppt1  (Creative BioMart)

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Recombinant Human PPT1 , Creative Biomart , Cat#PPT1-367H.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: Autophagy

Article Title: GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

doi: 10.1080/15548627.2021.1988357

Figure Lengend Snippet: Figure 5. GNS561 targets PPT1. (A) Nano differential scanning fluorimetry assays comparing GNS561 + PPT1 and HCQ + PPT1 against the apo-PPT1 ligand. Data represent the mean first derivative values (solid lines) ± SEM (shaded areas) of two experiments. SD from the mean is indicated by the light-color shading around the mean-line. Tm were determined by detecting the maximum of the first derivative of the fluorescence ratios. ΔTm values of each compound condition were determined by subtracting average Tm of PPT1 (in the respective buffer) by the average Tm of the respective compound condition. (B) PPT1 enzymatic activity of HepG2 cells treated with GNS561 for 3 h. HCQ and HDSF were used as positive controls. The results were compared to the diluent of GNS561 (control condition). (C) Representative immunoblotting of LC3-II in HepG2 cells treated with GNS561 for 16 h in the presence or absence of NtBuHA (8 mM). GAPDH was used as a loading control. Fold changes of normalized LC3-II level were calculated against the control condition (diluent of GNS561 + diluent of NtBuHA). (D) Cell viability percent against the control condition (diluent of GNS561 + diluent of NtBuHA) after 24 h of treatment with GNS561 in the presence or absence of NtBuHA (8 mM). (E) Fold change of normalized LC3-II (norm LC3-II) level were calculated against the control condition (diluent of GNS561) in HepG2 cells WT or siRNA-PPT1 treated by GNS561 for 24 h. GAPDH was used as a loading control. (F) Ratio of norm LC3-II between siRNA-PPT1 and WT HepG2 cells treated by GNS561 for 24 h. (G) Cell viability percent against the control condition (diluent of GNS561) after 24 h of treatment with GNS561 of WT and siRNA-PPT1 HepG2 cells. (H) Staining of lysosomes (LAMP2, green), MTOR (red) and nucleus (4′,6-diamidino-2-phenylindole [DAPI], blue) after treatment with GNS561 and two positive controls, EAD1 and HCQ, for 16 h. Pearson correlation coefficient between MTOR and LAMP2 was represented using scatter dot plot representation. In (B), (C), (D), (E), (F) and (G), data represent the mean + SEM. For comparison, Student t-test was used for (C), (D), (E), and (G) and one-way ANOVA with Dunnett’s post hoc analysis was performed for (B), (F) and (H). For all studies except (A), n ≥ 3 biological replicates. *represents significant difference, at least p < 0.05.

Article Snippet: Mammalian Cell Lysis Buffer (GE Healthcare, 28–941279), 4-methylumbelliferyl-β-D-6-thiopalmitoylglucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmpleteTM Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

Techniques: Nano Differential Scanning Fluorimetry, Fluorescence, Activity Assay, Control, Western Blot, Staining, Comparison

Journal: Autophagy

Article Title: GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

doi: 10.1080/15548627.2021.1988357

Figure Lengend Snippet: Figure 7. Schematic representation of molecular and cellular mechanisms involved in the antitumoral activity of GNS561. (A) Schematic illustration showing the stages of untreated tumor progression where autophagy activation and overexpression of PPT1 have been singled out in cell survival and tumor growth. (B) GNS561 compound localizes in lysosomes where it binds and inhibits PPT1 resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, autophagic flux inhibition, alters location of MTOR and leads to lysosomal membrane permeabilization. Finally, all these events induce caspase activation and tumor cell apoptosis.

Article Snippet: Mammalian Cell Lysis Buffer (GE Healthcare, 28–941279), 4-methylumbelliferyl-β-D-6-thiopalmitoylglucoside (Moscerdam, EM06650), Z-VAD-FMK (Bio Techne, FMK001), sorafenib (Santa Cruz Biotechnology, Sc357,801), hexadecanesulfonyl fluoride (HDSF; Santa Cruz Biotechnology, sc-221,708), human PPT1 (OriGene Technologies, TP721098), DC661 (Vagdevi Innoscience), Triton X-100 (Dutscher, 091584B) and cOmpleteTM Protease Inhibitor Cocktail (Roche, 4,693,132,001) were used.

Techniques: Activity Assay, Activation Assay, Over Expression, Inhibition, Membrane

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: 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 PPT1 E8/E1 fibroblasts and NSCs, and also there is no TPP1 expression detected in TPP1 E4/E6 and TPP1 E4/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

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Western Blot, Expressing, Staining

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: Effect of DT on reducing enlarged lysosomes in NCL patient NSCs. DT dose-dependently reduced the LysoTracker staining ( a ) in NCL NSCs. The quantitative analysis of LysoTracker fluorescence ( b ) revealed that clearance of enlarged lysosomes ranged from 22.8% in the TPP1 E4/E6 NSCs to 33.8% in the PPT1 E8/E1 NSCs after the treatment with 20 μM DT. The images were taken with 40X objective lens. Data are displayed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: Staining, Fluorescence

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: Hydroxypropyl-β-cyclodextrin (HPBCD) ameliorated enlarged lysosomes in NCL NSCs. a Representative image of HPBCD’s effect on reducing LysoTracker staining in NCL NSCs. The maximum reduction of enlarged lysosomes ranged from 31% in TPP1 E4/IVS5 NSCs to 47% in PPT1 E8/E1 NSCs after 1 mM HPBCD treatment ( b ). The images were taken with 40X objective lens. Data are displayed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: Staining

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: 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 PPT1 E8/E1 , TPP1 E4/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 PPT1 E8/E1 and TPP1 E4/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 PPT1 E8/E1 fibroblast were weaker than WT, but the expressions of subunit c increased in TPP1 E4/E6 and TPP1 E4/IVS5 fibroblast compared to WT ( b ). It showed that subunit c expression was decreased by 64% in PPT1 E8/E1 fibroblast, and increased 1.5-fold in both TPP1 E4/E6 and TPP1 E4/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 PPT1 E8/E1 NSCs than WT, but the expressions of subunit c were increased in TPP1 E4/E6 and TPP1 E4/IVS5 NSCs compared to WT ( d ). It showed that subunit c expression was decreased by 36% in TPP1 E4/E6 NSCs, and increased 1.2-fold in both TPP1 E4/E6 and TPP1 E4/IVS5 NSCs compared to WT ( e ). Data are displayed as mean ± SD. * P < 0.05, ** P < 0.01, compared to the WT control

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Marker, Fluorescence, Immunostaining, Recombinant, Staining, Western Blot

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: 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 PPT1 E8/E1 NSCs decreased by 75% ( a and b ). Moreover, it showed that subunit c expression in TPP1 E4/E6 NSCs and TPP1 E4/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

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: Expressing

Journal: Orphanet Journal of Rare Diseases

Article Title: Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

doi: 10.1186/s13023-018-0798-2

Figure Lengend Snippet: Summary of human cell lines used in the study

Article Snippet: Human recombinant PPT1 (14703-H08H) was purchased from Sino Biological (Beijing, China), and TPP1 (2237-SE-010) was ordered from R&D Systems (Minneapolis, MN).

Techniques: