Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is recruited to lipid droplets (LDs) in a GTPase-dependent manner in Hep3B cells . Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing ( A ) WT Rab5 (GFP-Rab5 WT), ( B ) constitutively active mutant (GFP-Rab5 Q79L), and ( C ) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red ) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green ) marks Rab5 localization. Inlays depict Rab5 localization around LD borders, indicated further by yellow arrows . D , quantification of Rab5–LD colocalization by the ratio of LD:cytoplasm GFP-Rab5 intensity. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The GFP-Rab5 was a gift from Marci Scidmore (Addgene plasmid #49888).

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: GFP-Rab5 plasmid , Addgene , 56417.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: GFP-Rab5 DN plasmid , Addgene , 28045.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: GFP-Rab5 Q79L mice exhibit motor deficits (A) Hindlimb clasping scores for GFP control mice (squares) and GFP-Rab5 Q79L mice (circles) at the indicated ages. (B) Number of falls in 2 min during the hanging wire behavioral assay for 3-month-old GFP and GFP-Rab5 Q79L mice. (C) Western blots of mouse brain lysates confirm expression of 3-month-old GFP and GFP-Rab5 Q79L mice. Upper panel: immunoblot using an anti-Rab5 antibody; lower panel: immunoblot using an anti-GFP antibody. Open arrow: GFP-Rab5 Q79L ; solid arrow: Rab5 or GFP. Cropped blots are shown in full in . (D) Quantification of Rab5 protein expression levels from (C). n = 9 and 10 mice in the GFP group and in the GFP-Rab5 Q79L group, respectively. All behavioral tests were repeated three times per mouse. Data are shown as the mean ± SEM. ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001; two-way ANOVA (A) and unpaired two-tailed t test (B and D).

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: Control, Behavioral Assay, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: Rab5 Q79L induces endosomal enlargement and abnormal lysosome morphology, recapitulating endolysosomal dysfunction (A) Immunofluorescence for GFP (green) and early endosome antigen 1 (EEA1; red), an early endosome marker, in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. DAPI marks nuclei in blue. Scale bar is 20 μm. (B) Approximate size of endosomes calculated by measuring the area of EEA1-positive vacuoles from the immunofluorescence images in A n = 5 mice per group. (C) Immunohistochemistry for the lysosome marker cathepsin D (CatD) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. Arrows: abnormal CatD puncta. Slides were stained with hematoxylin. Scale bar is 60 μm. (D) The percentage of cells with enlarged CatD-positive puncta is quantified; n = 5 mice per group. (E and F) (E) Immunofluorescence for GFP (green) and CatD (red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice with corresponding quantification (F). DAPI marks nuclei in blue. Scale bar is 10 μm. (G) Electron microscopy of the brains of 3-month-old GFP and GFP-Rab5 Q79L mice. Scale bars on left, middle, and right columns are 5, 1, and 500 nm, respectively. Black arrows indicate enlarged endolysosomes. Data are shown as the mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired two-tailed t test.

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: Immunofluorescence, Marker, Immunohistochemistry, Staining, Electron Microscopy, Two Tailed Test

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: GFP-Rab5 Q79L mice exhibit defects in proteostasis (A) Immunohistochemistry for ubiquitin in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. Slides were stained with hematoxylin. Scale bar is 60 μm. (B) The percentage of cells with ubiquitin aggregates is quantified; n = 5 mice per group. (C and D) (C) Immunofluorescence for GFP (green) and ubiquitin (red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice with corresponding quantification (D). DAPI marks nuclei in blue. Scale bar is 10 μm. (E) Immunohistochemistry for p62 in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. Slides were stained with hematoxylin. Scale bar is 60 μm. (F) The percentage of cells with p62 aggregates is quantified; n = 5 per group. (G and H) (G) Immunofluorescence for GFP (green) and p62 (red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice with corresponding quantification (H). DAPI marks nuclei in blue. Scale bar is 10 μm. Data are shown as the mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired two-tailed t test.

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: Immunohistochemistry, Ubiquitin Proteomics, Staining, Immunofluorescence, Two Tailed Test

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: Rab5 Q79L induces neurodegeneration and neuroinflammation in vivo (A–F) Representative images and quantification of immunohistochemistry for the neuronal (NeuN, A and B), astrocytic (GFAP, C and D), and microglial (Iba1, E and F) markers in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. Slides were stained with hematoxylin. Scale bars in (A) are 300 μm (NeuN). Scale bars in (C) and (E) are 60 μm (GFAP and Iba1). n = 6 mice per group in panels (B), (D), and (F). Data are shown as the mean ± SEM. ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001, unpaired two-tailed t test.

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: In Vivo, Immunohistochemistry, Staining, Two Tailed Test

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: Rab5 Q79L induces TDP-43 proteinopathy but not TDP-43 mislocalization nor loss of cryptic splicing in vivo (A and B) (A) Immunohistochemistry for TDP-43 phosphorylated at serines 409 and 410 (pTDP-43) in the cortex and thalamus of 3-month-old GFP-Rab5 Q79L mice, with ordinal score (B). Slides were stained with hematoxylin. Scale bars are 60 μm. (C) Immunofluorescence for GFP (green) and pTDP-43 (red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. DAPI marks nuclei in blue. Scale bar is 10 μm. (D) Immunohistochemistry for total TDP-43 in the cortex and thalamus of 3-month-old GFP-Rab5 Q79L mice. (E) Cryptic exon inclusion within TDP-43 target transcripts Sortilin1 and Ap3b2 between 3-month-old GFP and GFP-Rab5 Q79L mice. n = 6 mice per group. Data are shown as the mean ± SEM. ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001; ns, not significant; unpaired two-tailed t test.

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: In Vivo, Immunohistochemistry, Staining, Immunofluorescence, Two Tailed Test

Journal: iScience

Article Title: Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo

doi: 10.1016/j.isci.2025.113460

Figure Lengend Snippet: Rab5 Q79L induces morphological alterations in the nuclear envelope and nuclear pore complex (A) Immunofluorescence for GFP (green) and Lamin B1 (red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. DAPI marks nuclei in blue. Scale bar is 10 μm. (B) The percentage of cells with abnormal Lamin B1 staining. n = 3 mice per group. (C) Immunofluorescence for GFP (green) and nuclear pore complex (NPC, red) in the cortex of 3-month-old GFP and GFP-Rab5 Q79L mice. DAPI marks nuclei in blue. Scale bar is 10 μm. (D) The percentage of cells with abnormal NPC staining. n = 3 mice per group. Data are shown as the mean ± SEM. ∗∗∗∗ p < 0.0001 and ∗∗ p < 0.01; unpaired two-tailed t test.

Article Snippet: Accordingly, GFP-Rab5 Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: RAB5 NUCLEOTIDE BINDING PROMOTES β-OXIDATION TO FUEL HEPATOCELLULAR CARCINOMA CELL PROLIFERATION

doi: 10.1101/2025.08.20.670915

Figure Lengend Snippet: Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing (A) wild-type Rab5 (GFP-Rab5 WT), (B) constitutively active mutant (GFP-Rab5 Q79L), and (C) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green) marks Rab5 localization. Each panel contains two high-magnification insets (i and ii): inset i and ii zooms in on Rab5 puncta at LD borders, while below each of this insets shows the grayscale GFP channel alone, highlighting Rab5 signal independent of ORO. In Panel A, yellow arrows indicate discrete punctate Rab5 structures in close proximity to ORO-stained LDs, representing moderate LD association by wild-type Rab5. In Panel B, yellow arrows highlight continuous GFP-Rab5 rings surrounding smaller LDs, showing enhanced recruitment of the GTP-locked Rab5 Q79L to LD surfaces. In Panel C, yellow arrows mark areas where LDs lack adjacent Rab5 signal, and the GFP-Rab5 S34N mutant remains diffusely distributed throughout the cytoplasm, confirming a failure in membrane targeting due to its GDP-bound inactive state. (D) Quantification of Rab5-LD co-localization. The LD/Cytoplasm Intensity Ratio was calculated for each mutant by dividing the average GFP-Rab5 intensity at LD surfaces by the cytoplasmic GFP-Rab5 intensity. Bars represent mean ± SD from 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. **p<0.01 and ***p<0.001. Scale bars: main images, 10 µm; insets, 2 µm.

Article Snippet: The constitutively active GFP-Rab5 (Q79L) and dominant-negative GFP-Rab5 (S34N) constructs were gifted from Dr. Sergio Grinstein (Addgene plasmids #35140 and #35141, respectively).

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence, Membrane