Fig 1: AT-1 sTg and AT-1S113R/+ display alterations in the CD-M6PR, CI-M6PR, and lysosomal networks. (a) Representative electron microscopy of MEFs from WT, AT-1 sTg, and AT-1S113R/+ (scale bar, 2 µm). (b) Lysosomal morphology in primary-cultured MEFs using LysoTracker stain (20 × scale bar, 30 µm; 90 × scale bar, 15 µm) and quantified using Imaris reconstruction of surface area and number of puncta (n = 30 WT; n = 30 AT-1 sTg; n = 30 AT-1S113R/+). (c) Peroxisome morphology in primary-cultured MEFs using Peroxisome CellLight stain (scale bar, 10 µm) and quantified using Imaris reconstruction of number of puncta (n = 8 WT; n = 8 AT-1 sTg; n = 7 AT-1S113R/+). (d) Representative CD-M6PR in primary cultured MEFs using CD-M6PR antibody (scale bar, 3 µm). Surface area was quantified using Imaris reconstruction (n = 13 WT; n = 22 AT-1 sTg). (e) Representative CI-M6PR in primary cultured MEFs using CI-M6PR antibody (scale bar, 3 µm). Surface area was quantified using Imaris reconstruction (n = 16 WT; n = 12 AT-1 sTg). Two-way ANOVA (b,c). Welchs’ t test (d,e). **P < 0.005; #P < 0.0001.
Fig 2: Overexpression of Lamp2 alleviates dysfunction of the cathepsin trafficking induced by OGD treatment. (A,B) Double staining of cathepsins and Lamp1 to determine the trafficking of cathepsin D (A) and cathepsin B (B). Scale bar, 10 µm. (C,D) Quantitative analysis of (A,B). Mean ± SEM (n = 3). **P < 0.01 versus the normoxia + NC group and #P < 0.05, ##P < 0.01 versus the OGD + NC group. (E,F) Western blotting was performed to determine the protein levels of CD-M6PR and Cl-M6PR with Lamp2 overexpression (E). Quantitative analysis (F) is presented as the mean ± SEM (n = 3). **P < 0.01 versus the normoxia + NC group, ##P < 0.01 versus the OGD + NC group.
Fig 3: GCase-BS lysosomal mode of action in vitro.a Immunolabeling of hBS and colocalisation with LAMP1 upon acute treatment with various constructs. White arrows indicate some colocalising spots. Colocalising hBS spots were quantified and normalised to total amount of LAMP1 spots. n = 3. b Immunolabeling of hGCase and colocalisation with LAMP1 upon acute treatment with various constructs. White arrows indicate some colocalising spots. Colocalising GCase spots were quantified and normalised to total amount LAMP1 spots. n = 3. c Total GCase activity in GBA-deficient TfR WT and TfR KO neuroblastoma lines as a measure of cellular uptake after 2 h of treatment. Data was normalised to GB+/+ cells. n = 3. d GlcSph measurement in GBA-deficient TfR WT and TfR KO neuroblastoma lines upon 48 h of treatment. Data was normalised to respective GBA-/- cells. n = 3. e Total GCase activity in GBA-deficient M6PR-CI WT and M6PR-CI KO neuroblastoma lines as a measure of cellular uptake after 2 h of treatment. Data was normalised to GBA+/+ cells. n = 3. f GlcSph measurement in GBA-deficient M6PR-CI WT and M6PR-CI KO neuroblastoma lines upon 48 h of treatment. Data was normalised to respective GBA-/- cells. n = 3. g Total GCase activity in GBA-deficient M6PR-CD WT and M6PR-CD KO neuroblastoma lines as a measure of cellular uptake after 2 h of treatment. Data was normalised to GB+/+ cells. n = 3. h GlcSph measurement in GBA-deficient M6PR-CD WT and M6PR-CD KO neuroblastoma lines upon 48 h of treatment. Data was normalised to respective GBA-/- cells. n = 3. Bar graphs represent group means + SEM. Each data point represents an independent measurement. Data were analysed by Student’s two-tailed t-test comparing WT and KO of each receptor for each treatment. *p < 0.05; **p < 0.01; ***p < 0.001. If not stated otherwise, n = number of independent measurements. Source data are provided as a Source Data file.
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