Fig 1: BDNF induces a wave of TrkB signaling endosomes.(A) Corticostriatal connections form in microfluidic chambers in vitro. Kymographs, which show vesicle movement in both directions, were acquired at DIV10 to DIV12 when the neurons reach a mature stage, as shown by staining with synaptophysin I (SypI) and postsynaptic density 95 (PSD95). Scale bars, 20 µm (for kymograph) and 2 µm (for SypI/PSD95). LV, lentiviral vector. (B) Kymographs show movement of TrkB-mCherry vesicles before and 5 min after BDNF infusion (50 ng/ml). (C) BDNF treatment led vesicles to change their direction of movement from anterograde to retrograde. Kruskal-Wallis test, Dunn’s multiple comparisons test, *P < 0.05 and **P < 0.01; n = 30 to 40 axons from five independent experiments. (D) Analysis of axonal TrkB-mCherry vesicle transport from T0 and every 2.5 min thereafter. Each dot represents the average number of vesicles observed for each 100 µm of a given axon (n = 30 to 40 axons from five independent experiments). Left: Number of TrkB-containing anterograde vesicles remained fairly steady over time. Right: Number of retrograde vesicles peaked at T5. Kruskal-Wallis test, Dunn’s multiple comparisons test, *P < 0.05 and **P < 0.01. (E) After adding BDNF labeled with QDs (QDs-BDNF) to the synaptic compartment (top), we recorded the movement of TrkB-GFP and QDs-BDNF with dual acquisition at 5 and 75 min (bottom). Scale bars, 10 µm. (F) Distal TrkB-mCherry endosomes colocalized with Rab5-YFP, which is present on early endosomes; BDNF treatment in the synaptic compartment increased colocalization of TrkB endosomes with Rab5-positive vesicles. Unpaired two-tailed Student’s t test, **P < 0.01; n = 30 to 32 axons from three independent experiments. Scale bars, 2 µm.
Fig 2: Rab5 is upregulated in glioma tissues and cell lines. (A) Analysis of overall survival of patients with glioma and different levels of Rab5 expression. (B) The expression level of Rab5 in glioma tissues and pair-matched normal tissues was detected by RT-qPCR. Relative expression of Rab5 in glioma cells was measured by (C) RT-qPCR and (D) western blot analysis. *P≤0.05 vs. HNA. RT-qPCR, reverse transcription-quantitative PCR.
Fig 3: Rab5 is associated with the expression of cyclin E in glioma cells. (A-C) The expression of cell cycle-associated proteins was measured in (A) U87-MG, (B) SHG-44 and (C) A172 glioma cells by RT-qPCR. (D) The level of cyclin E in glioma cells was detected by western blot analysis. (E) Correlation analysis between Rab5 and cyclin E expression assessed by RT-qPCR. *P≤0.05 vs. NC-Overex or Rab-Ko-NC. RT-qPCR, reverse transcription-quantitative PCR; CDK, cyclin-dependent kinase; Ko, knockout; overex, overexpression; NC, negative control.
Fig 4: Endocytosed HIV-1 Env traffics through Rab5+ early endosomes and into Rab14+ compartments. (A to E) HIV-1-infected CEM-A T cells were pulsed with probes for Env and transferrin (Tfn) for 15 min at 4°C, then chased at 37°C, fixed, and imaged by confocal fluorescence microscopy. Representative confocal slices are shown. All scale bars 5 µm. White arrows indicate examples of colocalization, while red arrows indicate examples of noncolocalization. (A) mRuby3-Rab5A KI CEM-A cells were infected, pulse-labeled with anti-Env Fab b12-AF647 and Tfn-AF488, and chased for 1 h. Env and Tfn partially colocalized with Rab5A. (B) Wild-type (WT) CEM-A cells were infected with HIV-1 virus bearing a Rab5A(Q79L) expression cassette in replacement of the nef open reading frame, expressing GFP-Rab5A(Q79L) in place of HIV-1 Nef protein. The cells were pulse-labeled with anti-Env Fab b12-Atto565 and Tfn-AF647, and chased for 20 min. Both Env and Tfn accumulated in Rab5A(Q79L)-induced swollen EEs. (C) HIV-1-infected GFP-Rab11A KI CEM-A cells were pulse-labeled with anti-Env Fab b12-AF647 and Tfn-AF555, and chased for 1 h. Env showed little overlap with Rab11, and Tfn overlapped partially with Rab11, but Tfn colocalized more so with Env. (D) CEM-A cells stably overexpressing high levels of TagRFP-Rab11A were treated as in panel C. Tfn accumulated in the swollen ERC induced by Rab11 overexpression, while Env did not. (E) GFP-Rab14 KI CEM-A cells were treated as in (C). Env and Tfn colocalized significantly with Rab14. (F) Fractional overlap between probes in the assays represented in panels A, C, and E; n = 40 infected cells per sample. “Env w/Rab5” as example denotes the percentage of Env-positive pixels that were also positive for Rab5. Bars represent means, error bars represent SE, and points represent the values for individual cells. Statistical significance was assessed by Brown-Forsythe and Welch ANOVA tests and Dunnett’s T3 multiple-comparison test. n.s., P > 0.05, *, P = 0.05, **, P = 0.01, ***, P = 0.001, ****, P = 0.0001.
Fig 5: Overexpression of Rab5 promotes proliferation, migration and invasion of glioma cells. (A) The expression of Rab5 was effectively increased by the Rab5 overexpression plasmid. (B-D) Proliferation rate of (B) U87-MG, (C) SHG-44 and (D) A172 glioma cells was measured by CCK-8 assay. (E-H) Migration and invasion ability were assessed by Transwell assays. Magnification, ×40. *P≤0.05 vs. NC-Overex. NC, negative control; overex, overexpression.
Supplier Page from Abcam for Anti-Rab5 antibody [EPR21801]