Fig 1: RABEP2 does not associate with Rab5, early endosomes or macropinosomes. (A) HEK293 cells were transfected with Flag-tagged-Wild-type (WT), Ser204Glu (204E) or Ser200Ala/Ser204Ala (DBL) RABEP2 expression vectors. Cell lysates were prepared 24 h later and 0.5 mg subjected to immunoprecipitation with anti-FLAG. The entire immunoprecipitate (IP), and 7.5% of the remaining supernatants (SN), underwent SDS-PAGE and transfer to nitrocellulose. The membrane was cut into three by molecular mass and the upper section probed with anti-total RABEP2, the middle section with anti-GSK3 and the lower with anti-rab5 antibodies (original images of membrane sections are provided in Supplementary Information). (B) H9C2 cells were co-stained for endogenous Rab5 (green) and endogenous RABEP2 (red) along with DAPI staining of nuclei. Channels are shown individually and after merging images. The average Pearson’s coefficient for 26 different images was 0.219 ± 0.143, suggesting little, if any, co-localisation. (C) GFP-tagged WT RABEP2 (green) was overexpressed in HEK293 cells and cells loaded with Transferrin-Alexa Fluor594 (red) to label early endosomes. Channels are shown individually and after merging images. (D) To image F-actin in the cytoskeleton H9C2 cells were incubated for 5 mins in PBS containing 0.33 uM (final) AlexaFluor488 Phalloidin (Green), and counterstained for RABEP2 (red). Coverslips were visualised using a Leica SP5 laser scanning confocal microscope. Two representative images are provided. (E) H9C2 cardiomyoblasts were serum starved for 3 hours before incubation with DMSO vehicle, PMA or CT99021 for 1 hr as indicated. FITC-dextran (green) was added to the cells to label macropinosomes, endogenous RABEP2 was visualised by immunofluorescence (red), and nuclei were stained using DAPI. Two representative images for each condition are shown. The data indicates that GSK3 inhibition (CT) does not cause internalisation of FITC-dextran into macropinosomes, while the PKC inhibitor (PMA) is a known inducer of macropinocytosis (arrowheads indicate macropinosomes). These very distinct intracellular structures do not co-stain with RABEP2 implying that RABEP2 is not localised onto macropinosomes.
Fig 2: Effects of Rab5 and Rab7 knockdown on Zika virus (ZIKV) infection. (A) The knockdown efficiency of Rab5 or Rab7 was determined by Western blotting. T98G cells were transfected with either siControl, siRab5, or siRab7 for 48 h. Cell lysates were reacted with an anti-Rab5 antibody or an anti-Rab7 antibody specific for the indicated proteins. β-actin was used as an internal loading control. One representative experiment out of three is shown. (B) Rab5 or Rab7 depletion reduced ZIKV propagation. T98G cells transfected with the indicated siRNAs were infected with ZIKV at a multiplicity of infection (MOI) of 0.5 and incubated for 48 h to allow virus propagation. The infected cells were lysed to quantitate viral RNA copy numbers by RT-qPCR. (C) At 48 h post-infection, the titers of supernatant viruses were determined by plaque assay. (D) ZIKV localized in Rab5- and Rab7-postitive endosomes. T98G cells were transfected with EGFP-tagged Rab5 or Rab7 for 24 h, followed by infection with ZIKV at an MOI of 10 at 4°C for 1 h and then shifted to 37°C. At 24 h post-infection, the cells were fixed and stained with an anti-ZIKV envelop antibody (red). Nuclei were stained with DAPI (blue). One representative experiment out of three is shown (A). The immunofluorescence was examined under a confocal microscope. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm (D). The data shown are the mean ± SD of three independent experiments (B,C). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: Enlarged compartments in ClC-7Y713F cells are of late endosomal/lysosomal nature. (A) Representative images of HeLa cells co-transfected with rClC-7Y713F and RFP-tagged Ostm1 (red) and immune-stained (green) with antibodies against early endosomes (Rab5, EEA1), late endosomes (Rab7a, LBPA) or lysosomes (CD63, LAMP1). Nuclei were stained with DAPI (blue). Scale bar, 25 µm. White arrowheads in the lowest panel, which displays magnifications of marked squares in images above, show enlarged vesicles positive for late endosomal and lysosomal markers. Single channels of insets (EEA1, LBPA and LAMP1) and equivalent areas (Rab5, Rab7a and CD63) are shown in Figure S1. (B) Immuno-EM of HeLa cells co-transfected with ClC-7 (WT or Y713F) and Ostm1-GFP and stained with anti-LAMP1 and anti-GFP. The enlarged compartments stain positive for both GFP (15 nm gold, exemplarily shown with filled arrowheads) and LAMP1 (10 nm gold, empty arrowheads) reinforcing their late endosomal/lysosomal nature. G—Golgi apparatus; M—mitochondria; N—nucleus. Scale bar, 200 nm.
Fig 4: Increased escape of tau seeds from the endo-lysosomal system facilitates seeded aggregation. a, confocal images of HeLa cells expressing EGFP, Rab5 S34N-EGFP, or Rab7 T22N-EGFP (green) treated with DyLight-labeled aggregated P301S tau (red) for 3 h and incubated for a further 24 h and immunostaining with anti–galectin-8 (magenta). Arrows indicate co-localization of tau seed–containing vesicles with galectin-8. Scale bars, 10 μm. b, quantification of the percentage of cells with galectin-8–positive tau seed–containing vesicles. >100 cells counted/coverslip. c, flow cytometry fluorescence measurements of HeLa cells expressing EGFP, Rab5 S34N-EGFP, or Rab7 T22N-EGFP treated with DyLight-labeled aggregated P301S tau for 30 min at 37 °C. 10,000 cells/condition. d, representative Western blot with HT7 of the sarkosyl-insoluble fractions of 293T cells expressing P301S 1N4R tau and EGFP (control), Rab5 S34N-EGFP, or Rab7 T22N-EGFP treated with aggregated P301S tau for 3 h, followed by 48 h of growth. GAPDH was used as a loading control. e, densitometric analysis of HT7 blots of the sarkosyl-insoluble fractions from d. f, representative Western blot with HT7 of total lysates and sarkosyl-insoluble fractions of 293T cells expressing P301S tau treated with aggregated P301S tau for 3 h, followed by 48-h incubation with bafilomycin A1. GAPDH was used as a loading control. g, densitometric analysis of HT7 blots of total lysates (squares) and sarkosyl-insoluble (triangles) fractions from f. The results in b, c, e, and g are the means ± S.E. n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA).
Fig 5: Cdt treatment does not alter levels of intracellular trafficking proteins, EEA1, Rab5, Rab7, and LAMP1. THP-1 macrophages treated with CdtWT, CdtR117A, or control (untreated). (A) Cells were challenged with opsonized latex beads for 30 minutes and lysates collected for western blot as in Methods. (B) Quantified data represent mean +/- STDEV of EEA1, Rab5, Rab7, and LAMP1 levels (n=3) as percent of control (untreated) and compared using Student’s t-test.
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