gfp rab7 wt  (Addgene inc)

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: The vector constructs expressing GFP-Rab5, GFP-Rab11, and mCherry-Rab7, GFP-Rab7-WT and GFP-Rab7-DN were purchased from Addgene. siRNA against HRS, ALIX and TSG101 were purchased from Santa Cruz Biotech.

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-Rab7 plasmid , Addgene , 61803.

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-Rab7 DN plasmid , Addgene , 12660.

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

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: GFP-Rab7 T22N , Addgene , RRID:Addgene_28048.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) A schematic overview of apicosome formation in hPSC and classification of stages. Pre-apicosome stage occurs first as multiple PODXL vesicles (green) accumulates peri-nuclearly. PODXL vesicle traffic and fusion lead to the recruitment of EZRIN and aPKCσ (red circle) and proto-apicosome formation. Mature apicosome is formed as the proto-apicosome grows in size and acquire additional apical characteristics (e.g., microvilli (green projections), primary cilium (yellow projection), high Ca 2+ concentration (not shown)). B-E) Representative confocal images of H9 hESC harvested at 3-(B,D) and 6-(C,E) hr timepoints, which were stained with indicated markers (B and C, EE and RE components; D and E, LE/lysosome components). Two additional representative cells are shown in (EE/RE) and S3A-D (LE/lysosome). Arrowheads indicate PODXL/WGA structures that contain RAB7; arrows indicate structures that contain PODXL, RAB7 and lysotracker; asterisks indicate highly enlarged LE/lysosome compartments labeled by RAB7, LAMP1 and/or lysotracker, which lack PODXL/WGA. F) A schematic summarizing molecular characteristics of the endo-lysosomal system during the earliest stages of apicosome formation. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Concentration Assay, Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: Stained with indicated markers. Selected markers are shown. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images. Arrowheads indicate RAB7-labeled PODXL/WGA vesicles; asterisks indicate highly enlarged LE/lysosome compartments labeled by RAB7 and/or LAMP1. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A-C) H9 hESC carrying DOX-inducible GFP-RAB35-WT transgenic construct were treated with DOX starting at 0-hr, harvested at indicated timepoints (3-hr, (A); 6-hr, (B), 20-hr, (C)), and were stained with indicated markers. Representative confocal images of these samples are shown. Arrowheads indicate RAB7-labeled PODXL vesicles. Asterisks in (A) indicate highly enlarged LE/lysosome compartments that lack PODXL. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Transgenic Assay, Construct, Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: (A) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC, black), RAB35 -KO (red), and RAB35 -KO + GFP-RAB35-WT without (green) or with (purple) DOX treatment (n = 100 cells in each of the three independent experiments in each background, statistical significance based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test (asterisks indicate statistical significance of selected pairwise comparisons, p < 0.05)). (B) Quantitation for the diameter at the widest point of PODXL negative LAMP1 + LE/lysosome vesicles in control (blue) as well as in RAB35 -KO (red) cells at 3-hr. Twenty pre-apicosome cells were counted for each background. Only nine PODXL negative LE/lysosome compartments were found in the KO background (among 20 total cells); twenty nine LE/lysosome compartments were found among 20 cells in the control background. Statistical significance (asterisk, p < 0.05) is based on Student’s t-test. (C,D) Representative confocal images of 20-hr control and RAB35 -KO cells stained with selected apical (PODXL, WGA, pERM), EE (RAB5, EEA1), RE (RAB11), LE/lysosome (RAB7, LAMP1) markers. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A,B) Representative confocal images of 3-hr control and RAB35 -KO cells stained with indicated markers (EE and RE compartments (A); LE/lysosome compartments (B)). Asterisks in (B) indicate highly enlarged LE/lysosomes that are labeled by RAB7, LAMP1 and/or lysotracker. C) Western blot analysis of 3-hr control, RAB35 -KO #1 and RAB35 -KO #2 cells, blotted for PODXL, RAB7 and β-actin. Three independent replicates are shown for each sample group. D,E) Quantitations for normalized levels of PODXL (D) and RAB7 (E), normalized against total protein, using samples in (C). One-way ANOVA analysis showed a statistical significance in D (PODXL, p < 0.05), but not in E (RAB7, p > 0.05); asterisks indicate a statistical significance based on Tuckey’s test (p < 0.05). F) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr control and RAB35 -KO cells (n = 18 cells per background, asterisk indicates a statistically significant difference based on Student’s t-test (p < 0.05)). Insets indicate magnified regions shown as merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control, Staining, Labeling, Western Blot, Quantitation Assay

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) Representative confocal images of RAB35 -KO cells carrying DOX-inducible GFP fused RAB35-WT,-CA (constitutively active, Q67L) and -DN (dominant negative, S22N) transgenic constructs treated with DOX for 20 hours, stained with pERM and PODXL. In the “no DOX” condition (top), DOX-untreated 20-hr RAB35 -KO + RAB35-WT cells were harvested at the 20-hr timepoint. Note that the expression of the RAB35-DN construct does not lead to single apicosome formation. B) Quantitations for 20-hr cells with more than one apicosome for indicated background (n = 100 cells in each of the three independent experiments in each background. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test (asterisks indicate statistical significance of selected pairwise comparisons, p < 0.05)). C-F) Representative optical section of RAB35 -KO cells carrying inducible GFP-RAB35-WT (DOX-untreated in (C); DOX-treated in (D)), -CA (E) and -DN (F) constructs, harvested at the 3-hr timepoint (with 3-hr DOX treatment) and stained with indicated markers. Note that, while seen with the expression of RAB35-WT or -CA, the expression of the RAB35-DN construct does not lead to the formation of highly enlarged RAB7/LAMP1 double positive LE/lysosomes. Asterisks indicate the highly enlarged LE/lysosome compartments (D,E). G) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr cells from indicated sample group (n = 18 cells per background; numbers indicate total counted vesicles). Number of the PODXL vesicles is reduced to levels similar to controls when the RAB35-WT or -CA construct is expressed in the RAB35 -KO background, which is not seen with the expression of the RAB35-DN. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Dominant Negative Mutation, Transgenic Assay, Construct, Staining, Expressing, Quantitation Assay

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: (A) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC), RAB35 -KO, RAB35 -KO + GFP-RAB35-WT without DOX treatment, as well as DOX-treated RAB35 -KO + GFP-RAB35-WT, RAB35 -KO + GFP-RAB35-CA and RAB35 -KO + GFP-RAB35-DN cells (n = 100 cells in each of the three independent experiments in each background). A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05)). (B) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC), RAB35 -KO, RAB35 -KO + GFP-RAB7-WT without DOX treatment, as well as DOX-treated RAB35 -KO + GFP-RAB7-WT, RAB35 -KO + GFP-RAB7-CA and RAB35 -KO + GFP-RAB7-DN cells (n = 100 cells in each of the three independent experiments in each background). A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05)).

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) Representative optical section of RAB35 -KO cells inducibly expressing GFP-RAB7-WT, -CA (Q67L) or -DN (S22N) construct at 20-hr (with 20-hr DOX treatment), stained with indicated markers. In the “no DOX” condition (top), DOX-untreated 20-hr RAB35 -KO + RAB7-WT cells were harvested at the 20-hr timepoint. RAB7-WT or -CA expression leads to single apicosome formation while several apicosomes are still seen with RAB7-DN expression. B) Quantitation for the percentage of cells with more than one apicosome in the indicated genetic background (n = 100 cells in each of the three independent experiments in each background). Increase in RAB7 level or activity leads to reduced multi-apicosome formation. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). C-F) Representative confocal images of RAB35 -KO cells inducibly expressing GFP-RAB7-WT (minus DOX in (C), plus DOX in (D)), -CA (Q67L, (E)) or -DN (T22N, (F)) construct at 3-hr (with or without 3-hr DOX treatment), stained with indicated markers. Formation of then highly enlarged LE/lysosomal compartments (asterisks) is seen with RAB7-WT or -CA expression (D,E). G) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr cells from indicated sample group (n = 18 cells per background; numbers indicate total counted vesicles). Number of the PODXL vesicles is reduced to levels similar to controls when the RAB7-WT or -CA construct (but not -DN) is expressed in the RAB35 -KO background. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). H,I) Proposed model of apicosome formation at whole cell (H) and vesicle (I) levels. RAB35-RAB7 machinery promotes removal of excess PODXL vesicles, and contributes to single apicosome formation (H). At the earliest stages of apicosome formation (I), an EE/LE hybrid vesicle population is formed from a group of PODXL vesicles labeled with RAB35 and RAB5 by recruiting RAB7 and LAMP1. Subsequently, these hybrid vesicles shed RAB35 and RAB5, and are trafficked to an acidic LE/lysosomal compartment in a RAB35-RAB7-dependent manner, where PODXL is degraded. Much of the highly enlarged LE/lysosome compartments are resolved by the time the proto-apicosome is formed (top). Another group of RAB35/RAB5 PODXL vesicles removes RAB5 and recruits ERM proteins, which contribute to the formation of the proto-apicosome (bottom). Insets indicate magnified regions in the merged images. * = statistically significant; ns = statistically insignificant. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Expressing, Construct, Staining, Quantitation Assay, Activity Assay, Labeling