Fig 1: Recombinant and astrocyte-derived ApoE are internalized into the endosome–lysosome system in N2a cells.(A) Schematic overview of the 4 h treatment with 2.5 μg/ml recombinant ApoE3, ApoE4 or vehicle control in N2a cells. (B) Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue), and phalloidin (grey). Cells showing internalized human ApoE are indicated by arrows, and cells negative for ApoE are indicated by arrowheads. Scale bar is 40 μm. (C) Quantification of the percentage of ApoE-positive N2a cells within the entire culture. Recombinant ApoE4-treated N2a cells showed a significantly higher percentage of ApoE-positive cells (10.3% increase for ApoE4; t test, P = 0.024; number of cultures per treatment group: 5 [big data points in graph]; within each culture, 4–5 regions were analyzed [small data points in graph]). (D, E) Quantification of the number of ApoE puncta (D) and puncta area (E) per N2a cell. ApoE4 treatment significantly increased the median ApoE puncta number by 33.4% (mean puncta increase: 1.6) (Mann–Whitney test, P = 0.034) and showed a trend in the ApoE puncta area increase (by 27.5%) for ApoE4 compared with ApoE3 treatment (Mann–Whitney test, P = 0.0767); number of cultures per treatment group: 14, number of images taken within each culture: 1–7). (F) Higher magnification image of ApoE3- and ApoE4-treated N2a cells from Fig 1B. Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. (G) Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue), and phalloidin (grey). The scale bar represents 10 μm. (H) Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue), and phalloidin (grey). Scale bar is equal to 10 μm. (I) Quantification of Fig 1D, showing the co-localization levels (in percentages) of ApoE with LAMP1. After ApoE3 and ApoE4 treatment, on average 35.8% and 30.7% of ApoE, respectively, co-localized with LAMP1 (t test on difference between ApoE3- and ApoE4-treated N2a cells, P = 0.2744; number of cultures [big data points in graph]: 3; number of cells analyzed per culture [small data points in graph]: 5). (J) Quantification of ApoE-Rab7 co-localization of Fig 1F. 14.2% and 12.1% of the ApoE co-localized with Rab7 in N2a cells after ApoE3 and ApoE4 treatment, respectively (t test on difference between ApoE3- and ApoE4-treated N2a cells, P = 0.3392; number of cultures [big data points in graph]: 3; number of cultures analyzed per culture [small data points in graph]: 5). (K) Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. (L) Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI, and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. (M) Representative Western blot of secreted human ApoE proteins detected in astrocyte-conditioned media after culturing 48 h with ApoE KO, ApoE3 KI, and ApoE4 KI primary astrocytes. (N, O) Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (N) and to a lower extent also with Rab7 (red) (O) in ApoE3 and ApoE4 astrocyte-conditioned media–treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). (N, O) Arrows indicate ApoE puncta inside LAMP1- (N) and Rab7- (O) positive vesicles. Scale bars represent 5 μm. Data are expressed as mean ± SD. ns, nonsignificant, Rec ApoE, recombinant ApoE. See also Fig S1A–C.
Fig 2: EVs shuttle HMGB1 to AML-12 by transferrin-mediated endocytosis.a Representative confocal microscopy micrographs of AML-12 cells after co-culture with purified PKH67-EVs. b HMGB1-GFP EVs uptake occurred in vitro and in vivo. c The co-localization of internalized EVs and AML-12 endosomes (EEA1, Rab5, Rab7) or Lamp1. d The expression of transferrin receptor in EVs, the co-localization of internalized EVs with AML-12 transferrin. e The LPS-EVs absorbed by AML-12 cells. f Cytoplasmic HMGB1 in AML-12 cells. (**) P < 0.01 versus control group. (##) P < 0.01 versus EVs group.
Fig 3: fip5a and fip5b double mutants show severe microvilli and trafficking phenotypes. All following images are representative cross sections through the midgut region of 6 dpf larvae. (A–A′′′′) Electron micrographs showing wild-type siblings and fip5aCO35/CO35; fip5bCO40/CO40 zygotic mutant larvae. Arrows point to larger than 500 nm organelles, braces point out sparse microvilli and brackets mark terminal web or lack thereof in mutants. N indicates number of representative larvae out of total number of larvae analyzed. (B) Electron micrograph showing wild-type AB larva. (C) Quantitation of microvilli density. Each dot represents the number of microvilli per micron for a field of view across three wild-type and five mutant animals. (D) Immunohistochemistry on cross sections of wild-type and fip5aCO35; fip5bCO40 mutant larvae stained with Hoechst (blue), Phalloidin (red), and Rab7 (green). (E) Quantitation of Rab7-vesicle diameter. (F) Immunohistochemistry on cross sections of wild-type and fip5aCO35; fip5bCO40 mutant larvae stained with Hoechst (blue), Phalloidin (red) and Cytokeratin (green). (G) Ratio of fluorescence intensity of apical keratin to cytoplasmic keratin. Three separate animals for each condition were analyzed. All plots show mean±s.e.m. A t-test was used for Gaussian data and a Mann–Whitney test for all other statistics. ***P<0.0005.
Fig 4: Impaired Rab7 effector interactions in 22L-N2a cells.A, N2a and 22L-N2a cells were transfected with GFP-tagged WT-Rab7 and subjected to immune pull-down with anti-GFP nanobody-coupled agarose beads and subjected to immunoblotting with anti-RILP antibody. The quantitative analysis of the RILP signal normalized to the amount of GFP-tagged WT-Rab7 levels after stripping these membranes with methanol is also shown. B, immunofluorescence images depicting the localization of lysosomes, by probing with anti-Lamp1 antibody in N2a and 22L-N2a cells as visualized by the maximum intensity projections of the z-stack sections, obtained by confocal microscopy. All confocal images are representative of five fields of view from three independent experiments. Statistical significance was evaluated using the unpaired t test. The dashed and dotted lines in the violin plot depict the median and the lower and the upper quartiles of values, respectively. C, N2a and 22L-N2a cells were transfected with GFP-tagged WT-Rab7 and subjected to immune pull-down with anti-GFP nanobody-coupled agarose beads and subjected to immunoblotting, with anti-ubiquitin antibody. Quantitative analysis of the Ub-Rab7 signal normalized to the amount of GFP-tagged WT-Rab7 levels after stripping these membranes with methanol is also shown. Unpaired t test was used to analyze the statistical significance between different groups from three or more independent experiments, and the results are summarized in Table S1. The error bars indicate the standard deviation.
Fig 5: Colocalization of Rab 5, Rab 7 and Rab 11 in human spinal cord motor neurons in an ALS patient with C9ORF72-intronic repeat expansion mutation. (A) Immunohistochemistry of human postmortem spinal cord sections from a control individual without neurological disorders and a human ALS patient bearing C9ORF72-intronic mutation. Human postmortem spinal cord sections (5 µm) were immunostained with anti-Rab11, anti-Rab5 or anti-Rab7 (first column) antibodies and anti-C9ORF72 antibodies (second column). Merge (third column) indicates overlays of the fluorescent confocal images of C9ORF72 and each Rab. White arrow indicates areas of colocalization between C9ORF72 and Rabs in both control and ALS patient tissues. Scale bar: 20 μm, applied to all fields. (B) Quantification of motor neurons containing colocalized C9ORF72 and Rabs reveals an increased proportion of motor neurons in which C9ORF72 colocalized with Rab11 or Rab7 in tissues from an ALS patient bearing the C9ORF72 repeat expansion compared with a control patient. Fifty motor neuron cells were scored for each population. Data are represented as mean ± SEM; *P < 0.05, ALS versus control by unpaired t-test.
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