Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 1: EPI64A and EPI64B both localize to apical microvilli. (A) Schematic of EPI64A and EPI64B domains with percentage identities and the TBC/RabGAP arginine residue necessary for the RabGAP activity indicated. (B) Western blots with antibodies to EPI64B and tubulin of Jeg-3 cell lysates after treatment with the indicated siRNAs to EPI64B. (C) Western blot with antibodies to EPI64B on cell lysates from several cultured cell lines. (D) SIM microscopy of Jeg-3 cells transfected with GFP-EPI64A (top block of panels) or GFP-EPI64B (bottom block of panels). Cells were transfected to express GFP-tagged constructs (green) and stained for ezrin (red) and actin (blue). Arrows indicate the localization of GFP-EPI64A to the base of microvilli. Scale bars: top panels, 10 µm; bottom panels, 10 µm.(E) Quantitation of GFP- EPI64A and GFP-EPI64B to localize to microvilli in wild-type cells.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Residue, Activity Assay, Western Blot, Cell Culture, Microscopy, Transfection, Blocking Assay, Construct, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 2: EPI64A and EPI64B can localize to microvilli independently of the scaffolding protein EBP50 (A) Jeg-3 cells transfected with 3xFLAG-EBP50 were co-transfected with either GFP-EPI64A or GFP-EPI64B. The GFP-tagged proteins were immunoprecipitated with GFP-Trap beads and the immunoprecipitates blotted for FLAG and GFP. (B) Confocal imaging of microvillar localization of GFP-EPI-64A and GFP-EPI64A-LA, which cannot bind EBP50, in Jeg-3 cells. Scale bar 10 µm. (C) Western blot of cell lysates of Jeg-3 wild type, or CRISPR-modified EBP50 deletion cell line, blotted for ezrin, EBP50, and tubulin. Scale bar: 10 µm (D) Localization of ezrin and actin in Jeg-3 cells lacking endogenous EBP50. Scale bars: 10 µm. (E) Confocal imaging of GFP-EPI64A or GFP-EPI64B in Jeg-3 cells lacking EBP50. Scale bars: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Scaffolding, Transfection, Immunoprecipitation, Imaging, Western Blot, CRISPR, Modification

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 3: EPI64A contains a localization domain spanning its TBC domain. (A) Schematic of the EPI64 constructs used in B and C and summary of results shown in this figure (B) Confocal imaging of GFP-EPI64A-314-508, which contains the C-terminal -DTYL sequence, in Jeg-3 wild-type cells and Jeg-3 cells lacking EBP50. (C) Confocal images showing the localization of GFP-tagged deletion constructs of GFP-EPI64A. Scale bar 10 µm. (D) Immunolocalization of two HA-tagged constructs, the top one containing the minimal region that localizes to microvilli (HA-EPI64A-61-408) and the bottom one (HA-71-408) that does not localize. Scale bar: 10 µm. (E) GFP-trap pull down: Jeg-3 cells were transfected with either GFP or GFP-Arf6 together with the indicated HA-EPI64A constructs. The GFP or GFP-Arf6 were recovered and analyzed for the presence of the HA-EPI64A constructs by immunoblotting.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Construct, Imaging, Sequencing, Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 5: Jeg-3 cells lacking EPI64A and EPI64B lack microvilli (A) Western blot with antibodies to EPI64A, EPI64B, and tubulin on whole cell lysates of Jeg-3 cells genetically modified to lack EPI64A, EPI64B, or both proteins (DKO). (B) Confocal imaging showing localization of ezrin and actin in wild-type Jeg-3 cells and the single (A-KO, B-KO) and double knockout cells. Scale bar 10 µm. (C) Quantitation of the percentage of indicated cells stained for ezrin that express surface microvilli. Normal defined >50% coverage of the apical surface with microvilli. One-way analysis of variance gave the indicated p values. (D) EPI64A/B double knockout cells were transfected to express the indicated constructs and the percentage of ezrin-stained cells (total for either untransfected or GFP-expressing for transfected cells) that express normal apical microvilli. One-way analysis of variance gave the indicated p values. (E) Localization of tight junction ZO-1 in wild-type and knockout Jeg-3 cells. Scale bar: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Genetically Modified, Imaging, Double Knockout, Quantitation Assay, Staining, Transfection, Construct, Expressing, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 6: Dominant negative Rab8A and Rab35A can restore microvilli to EPI64A/B double knockout cells. (A) Wild-type Jeg-3 cells were transfected to express GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) determined that express apical microvilli. (B) Jeg-3 EPI64A/B double knockout cells were transfected with GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) expressing microvilli scored. One-way analysis of variance gave the indicated p values.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Dominant Negative Mutation, Double Knockout, Transfection, Expressing

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 7: Caco-2 cells lacking EPI64A and EPI64B have microvilli (A) Western blots of whole cell lysates from Caco-2 BBE1 wild-type, EPI64A, and EPI64B single knockout and double knockout cells blotted for EPI64A, EPI64B, and tubulin. (B) Confocal imaging showing localization of ezrin in the apical region of wild-type and knockout cells. Scale bar: 10 µm. (C) Localization of GFP-EPI64A and GFP-EPI64B expressed in double knockout cells. Scale bar 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Knock-Out, Double Knockout, Imaging

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 8: Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild–type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 µm. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180°). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Knock-Out, Double Knockout, Staining, Marker

Journal: International Journal of Molecular Medicine

Article Title: Transcription factor EB-mediated autophagy promotes dermal fibroblast differentiation and collagen production by regulating endoplasmic reticulum stress and autophagy-dependent secretion

doi: 10.3892/ijmm.2020.4814

Figure Lengend Snippet: TGF-β1-induced COL I secretion from fibroblasts is associated with an autophagy-based pathway. Fibroblasts were treated with 50 nmol/l TFEB-siRNA or NC-siRNA for 48 h and then with 10 ng/ml TGF-β1 for 48 h. (A) Fluorescence for colocalization of Rab8a with LAMP1 and COL I (scale bars, 10 or 30 µ m). (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for COL I and LAMP1. (D) Pearson's colocalization coefficient for COL I and Rab8a. (E) Pro-COL Iα1 secretion in the supernatant of fibroblasts was assessed by enzyme-linked immunosorbent assay. Results are presented as the mean ± SD (n=6). ** P<0.01 vs. control group; ## P<0.01 vs. TGF-β1 treatment group. TGF-β1, transforming growth factor-β1; TFEB, transcription factor EB; siRNA, small interfering RNA; NC, negative control; COL I, collagen I; Rab8a, Ras-related protein Rab-8A; LAMP1, lyso-some-associated membrane protein 1; ns, not significant compared with TGF-β1 treatment group.

Article Snippet: Subsequently, cells were incubated with the following primary antibodies overnight at 4°C: Anti-TFEB (1:50; cat. no. ab220695; Abcam), anti-LAMP1 (1:200; cat. no. ab62562; Abcam), anti-COL I (1:200; cat. no. ab6308; Abcam) and anti-Ras-related protein Rab-8A (Rab8a; 1:200; cat. no. ABIN6290618; Beijing 4A Biotech Co., Ltd.).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Negative Control

Journal: The Journal of Cell Biology

Article Title: EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

doi: 10.1083/jcb.201508086

Figure Lengend Snippet: EHBP1L1 is a novel Rab8-interacting protein. (A) Schematic representation of EHBP1L1. CH, calponin homology domain; CC, coiled-coil domain. Seven PXXP motifs are indicated by triangles. (B) Lysates from different mouse tissues were immunoblotted using an EHBP1L1 antibody. EHBP1L1 splicing isoforms based on the GenBank database are indicated by arrows. (C) A yeast two-hybrid assay between EHBP1L1 and the GTP-restricted forms of Rab proteins. The cells were grown on SC-LW plates, and five independent colonies were restreaked on QDO plates. (D) Yeast two-hybrid assay using EHBP1L1 and the GTP or GDP forms of Rab-8a, 8b, 10, and 13. (E) In vitro pull-down assay using recombinant GST-EHBP1L1-CC (top) or GST-EHBP1-CC (bottom) and the lysate from HEK293 cells expressing the FLAG-tagged GTP-form of Rab, as indicated. The transferred protein was stained with Ponceau S (PS) to detect GST-fusion proteins. The bound Rab proteins were detected by immunoblotting using a FLAG antibody. (F) HEK293 cell lysates coexpressing FLAG-tagged EHBP1L1 and EGFP-tagged Rab, or EGFP proteins were immunoprecipitated using a FLAG antibody.

Article Snippet: Experiments were performed 24 h after transfection. siRNA oligos for human EHBP1L1 (siRNA ID: SASI_Hs02_00320623) and Bin1 (siRNA ID: SASI_Hs01_00052359; Sigma-Aldrich) and Rab8a and Rab8b siRNA (SI02662254 and SI02662261; Qiagen) were transfected using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) as described in the manufacturer’s manual.

Techniques: Y2H Assay, In Vitro, Pull Down Assay, Recombinant, Expressing, Staining, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

doi: 10.1083/jcb.201508086

Figure Lengend Snippet: Rab8, EHBP1L1, and Bin1 stabilize each other on the tubules. (A) Immunofluorescence for EHBP1L1, Bin1, and Rab8a in HeLa cells. The enlarged views at the bottom show the overlap of the proteins on the tubular structures. Areas a and b are representative regions of the ERC depicting uniform intensity and heterogeneous intensity between the two proteins, respectively. Bars: (magnified views) 1 µm; (other views) 10 µm. (B) Correlation analysis comparing the distribution of EHBP1L1/Bin1 and Rab8/Bin1 in the whole cell (whole), the region positive for the two proteins (a), and the region positive for either protein (b). Data are mean ± SEM from at least 10 cells. ***, P < 0.001 relative to control; Student’s t test. (C) KD efficiency of Rab8, Bin1, and EHBP1L1 in HeLa cells. Lysates were analyzed by immunoblotting. Lamin B was used as a loading control. (D and E) HeLa cells transfected with siRNA for Rab8a and Rab8b, EHBP1L1, or Bin1 were stained with Rab8, Bin1, or EHBP1L1 antibody (green). Nuclei were indicated using DAPI (blue). Bar, 20 µm. (E) Quantification of the percentage of cells with tubular structures. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to control; Student’s t test.

Article Snippet: Experiments were performed 24 h after transfection. siRNA oligos for human EHBP1L1 (siRNA ID: SASI_Hs02_00320623) and Bin1 (siRNA ID: SASI_Hs01_00052359; Sigma-Aldrich) and Rab8a and Rab8b siRNA (SI02662254 and SI02662261; Qiagen) were transfected using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) as described in the manufacturer’s manual.

Techniques: Immunofluorescence, Western Blot, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

doi: 10.1083/jcb.201508086

Figure Lengend Snippet: Apical cargo protein is transported through the ERC. (A) Subcellular localization of Rab8, EHBP1L1, FLAG-tagged EHBP1L1, and Bin1 in EpH4 cells was examined using immunofluorescence microscopy. (B) Colocalization was analyzed between EHBP1L1 and Rab8a, Bin1 (A), Lamp2, sorting nexin 1 (SNX1), and internalized transferrin (Tf; Fig. S2 B). (C) EpH4 cells expressing VSV ex -FLAG-mCherry-GPI were cultured at 40°C and incubated for 60, 90, and 120 min at 32°C. The cells were fixed at each time point and stained using an EHBP1L1 antibody and Alexa Fluor 633 phalloidin. The insets show enlarged views. The arrowheads in the insets denote colocalization. Bars: (magnified views) 2 µm; (other views) 10 µm. (D) Colocalization was analyzed between EHBP1L1 and both VSV ex -FLAG-mCherry-GPI (apical cargo) and VSVG ts045 -GFP (basolateral cargo; Fig. S2 C) after 90-min chase. Data are mean ± SEM from 10 cells; P < 0.01.

Article Snippet: Experiments were performed 24 h after transfection. siRNA oligos for human EHBP1L1 (siRNA ID: SASI_Hs02_00320623) and Bin1 (siRNA ID: SASI_Hs01_00052359; Sigma-Aldrich) and Rab8a and Rab8b siRNA (SI02662254 and SI02662261; Qiagen) were transfected using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) as described in the manufacturer’s manual.

Techniques: Immunofluorescence, Microscopy, Expressing, Cell Culture, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

doi: 10.1083/jcb.201508086

Figure Lengend Snippet: EHBP1L1 and Bin1 depletion induces the accumulation of apical but not basolateral membrane proteins in lysosomes. (A) EHBP1L1 and Bin1 mRNA levels in mouse small intestine organoids were analyzed using semiquantitative RT-PCR. The level of GAPDH mRNA was used for standardization. (B) Immunostaining for Na + /K + ATPase (green) and DPP4 (red) in the mouse small intestine organoids. The nuclei were counterstained with DAPI (blue). The arrows indicate cytoplasmic DPP4 accumulation. Bar, 50 µm. (C) Magnified images of immunostaining for DPP4, Lamp2, and Na + /K + ATPase in the small intestine Bin1 or EHBP1L1 KD organoids or organoids obtained from Rab8a or Rab8a/8b DKO mice. Arrowheads, DPP4 accumulation in lysosomes; arrow, Na + /K + ATPase accumulation in lysosomes. Bar, 10 µm. (D) Immunostaining for DPP4 (red), Lamp2 (green), and Na + /K + ATPase (white) in mouse small intestine organoids treated with DMSO (control) or 40 µM dynasore for 4 d. Bar, 10 µm. (E) Electron micrographs of epithelial cells in small intestines from EHBP1L1 KO mice and wild-type mice. Bars: (top) 1 µm; (bottom) 10 µm. (F) Immunoblot for intestine lysate from a WT or an EHBP1L1 KO mouse using an EHBP1L1 antibody. (G) Working model for Rab8, EHBP1L1, Bin1, and dynamin at the ERC. Bin1 is represented as a dimer .

Article Snippet: Experiments were performed 24 h after transfection. siRNA oligos for human EHBP1L1 (siRNA ID: SASI_Hs02_00320623) and Bin1 (siRNA ID: SASI_Hs01_00052359; Sigma-Aldrich) and Rab8a and Rab8b siRNA (SI02662254 and SI02662261; Qiagen) were transfected using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) as described in the manufacturer’s manual.

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunostaining, Western Blot

Journal: Nature Communications

Article Title: Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening

doi: 10.1038/ncomms11166

Figure Lengend Snippet: ( a ) Saccharomyces cerevisiae L40 reporter strain was transformed with plasmids encoding GAD fused to PODXL cytoplasmic tail (aa 476–551) or GAD alone to examine interactions with LexA fused to Rab35 WT , Rab35 Q67L (GTP-bound mutant), Rab35 S22N (GDP-bound mutant), Rab11A Q70L (GTP-bound mutant), Rab6A Q72L (GTP-bound mutant), Rab8A Q67L (GTP-bound mutant) and Rab27A Q78L (GTP-bound mutant). Growth on a medium without histidine (−His) indicates an interaction with the corresponding proteins in this two-hybrid assay. ( b ) Recombinant GST or GST–PODXL (aa 476–551) immobilized on glutathione beads was incubated with recombinant 6xHis-tagged Rab35 WT , Rab11A WT or Rab6A WT , and loaded with either GDP or GTPγS. Top panel: Rab proteins directly bound to beads were detected by western blot with anti-6xHis antibodies. 5% of the Rab protein input are displayed in the first six lanes. Bottom panel: bead inputs in Ponceau staining. ( c ) Rab proteins were immunoprecipitated with anti-Flag antibodies (IP) from MDCK cells transfected with plasmids encoding 3xFlag-tagged Rab6A Q72L , Rab8A Q67L , Rab27A Q78L , Rab11 Q70L , Rab35 S22N , Rab35 WT or Rab35 Q67L . Flag-proteins and co-immunoprecipitated endogenous PODXL were detected by western blot using anti-PODXL antibodies (top panel) and anti-Flag antibodies (middle panel). Corresponding inputs (5% of total lysates) are displayed in the bottom panel.

Article Snippet: MDCK cells were transfected with either 3xFlag-tagged Rab35 WT , Rab35 S22N , Rab35 Q67L , Rab11A Q70L , Rab6A Q72L , Rab8A Q67L or Rab27A Q78L for 24 h using Amaxa.

Techniques: Transformation Assay, Mutagenesis, Two Hybrid Assay, Recombinant, Incubation, Western Blot, Staining, Immunoprecipitation, Transfection

Journal: Nature Communications

Article Title: Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening

doi: 10.1038/ncomms11166

Figure Lengend Snippet: ( a – h ) MDCK cells were transfected for 72 h with plasmids encoding either fluorescently tagged Rab35 Q67L or Rab35 S22N fused to the mitochondrial targeting signal of ActA (-Mito), as indicated. In the merged images, Rab35-Mito proteins are displayed in red, and PODXL, GFP-Crumbs3, GFP-Cdc42 or aPKC in green. ( i , j ) MDCK cells were treated with either control or PODXL siRNAs for 72 h, and co-transfected for 72 h with plasmids encoding mCherry-Rab11 WT and either GFP-Rab35 Q67L -Mito or GFP-Rab35 S22N -Mito, as indicated. ( k , l ) MDCK cells were co-transfected for 72 h with plasmids encoding mCherry-Rab35 Q67 -Mito and either GFP-PODXL WT or GFP-PODXL V496A Y500A. In the merged images, Rab35 Q67L -Mito is displayed in red, and PODXL WT or mutant in green. For a – l individual channels in grey levels and higher magnification of the regions delimited by a dash line are displayed, as indicated. Scale bars, 10 μm for unzoomed regions and 2 μm for zoomed regions. Arrowheads indicate examples of close-apposition vesicles (green) with mitochondrial Rab35 Q67L (red).

Article Snippet: MDCK cells were transfected with either 3xFlag-tagged Rab35 WT , Rab35 S22N , Rab35 Q67L , Rab11A Q70L , Rab6A Q72L , Rab8A Q67L or Rab27A Q78L for 24 h using Amaxa.

Techniques: Transfection, Control, Mutagenesis

Journal: Nature Communications

Article Title: Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening

doi: 10.1038/ncomms11166

Figure Lengend Snippet: ( a ) MDCK cells transfected with either GFP-Rab35 S22N -Mito or GFP-Rab35 Q67L -Mito were seeded into Matrigel for 24 h, cysts were fixed and stained for PODXL and F-actin (phalloidin). Merged images and single channels in grey levels are displayed, as indicated. Scale bar, 10 mm. Graphs: proportion of cysts with PODXL normally localized at the apical membrane (left graph) or with PODXL localized at the mitochondria (right graph) in each condition. Mean±s.d., N =3 independent experiments, 100–200 two-cell cysts analysed per condition. Two-way analysis of variance (ANOVA): *** P <0.001. ( b ) MDCK cells were transfected for 24 h with plasmids encoding either GFP-Rab35 Q67L -Mito or GFP-Rab35 S22N -Mito and seeded into Matrigel for 48 h. Cysts were then fixed and stained for PODXL (green), F-actin (phalloidin, red) and DNA (DAPI). Merged images and individual channels in grey levels are displayed. Scale bar, 10 mm. Graphs: normal cysts, cysts without lumen, inverted cysts and other abnormal cysts were quantified based on PODXL and Phalloidin staining. Two-way ANOVA: ** P <0.01; *** P <0.001; NS, not significant. Mean±s.d., N =3 independent experiments, >100 cysts analysed per condition.

Article Snippet: MDCK cells were transfected with either 3xFlag-tagged Rab35 WT , Rab35 S22N , Rab35 Q67L , Rab11A Q70L , Rab6A Q72L , Rab8A Q67L or Rab27A Q78L for 24 h using Amaxa.

Techniques: Transfection, Staining, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics augmented LPS-induced SAP secretion via the activation of Rab8a in vitro . (A) Western blot analysis demonstrated the Rab8a levels in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (B) Western blot analysis illustrated the efficient siRNA-mediated knockdown of Rab8a in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (C) NTA revealed significant decreases in vesicle levels from RAW264.7 cells with Rab8a knockdown in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (D) Western blot analysis demonstrated significant decreases in LC3-II levels in vesicles from LPS-stimulated RAW264.7 cells in the Rab8a knockdown groups compared with the control group. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the control; #p < 0.05 vs . the LPS control; **p < 0.05 vs . the mimics group without Rab8a knockdown; ***p < 0.05 vs . the inhibitors group without Rab8a knockdown, one-way ANOVA.

Article Snippet: On the other hand, RAW264.7 cells were also transfected by the RNAi of Rab8a (RiboBio, Guangzhu), pursuant to the manufacturer’s procedure.

Techniques: Activation Assay, In Vitro, Western Blot, Transfection, Knockdown, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics activate Rab8a by directly reducing PAK4 expression. (A) The predicted MiR-199a-3p binding site on the PAK4 3′-UTRs was determined using target prediction software. (B) Western blot analysis validated the PAK4 expression when cells were transfected with the MiR-199a-3p mimic/inhibitor. (C) Luciferase reporter plasmid assays with wild-type and mutated PAK4 plasmids co-transfected with MiR-199a-3p- or MiR-NC-packaged plasmids. Dual luciferase control vector plasmids acted as NCs. (D) Nanoparticle tracking analysis revealed significant increases in the level of vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (E) Western blot analysis demonstrated significant increases in LC3-II levels in vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (F) Western blot analysis revealed the effect of PF-3758309 on increasing Rab8a levels in RAW264.7 cells transfected with MiR-199a-3p mimics and inhibitors. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the plasmid-control group transfected with MiR-199a-3p plasmids; #p < 0.05 vs . the control group; **p < 0.05 vs . the mimics group without PF-3758309; ***p < 0.05 vs . the inhibitors group without PF-3758309, one-way ANOVA.

Article Snippet: On the other hand, RAW264.7 cells were also transfected by the RNAi of Rab8a (RiboBio, Guangzhu), pursuant to the manufacturer’s procedure.

Techniques: Expressing, Binding Assay, Software, Western Blot, Transfection, Luciferase, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A) Immunoblot analysis comparing lysates from RPE WT, tRFP-Rab8a, tRFP-Rab8a+GFP-Rab11a, tRFP-Rab8a+GFP-Rabin8, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab14 cells ± Dox probed with Rab8a antibody; RPE WT, tRFP-Rab8a+GFP-Rabin8, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab14, cells ± Dox probed with Rabin8 antibody; and RPE WT, tRFP-Rab8a+GFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab11a cells ± Dox and probed with Rab11a antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as a loading control. * corresponds to the endogenous protein detected in RPE WT cells and the arrows indicates the endogenous and exogenous levels in established fluorescent-tagged RPE cells. (B) Table showing densitometry of Rab11a, Rab8a, and Rabin8 proteins corresponding to arrows in (A) in ±Dox-treated cell lines compared to endogenous proteins in RPE WT cells corresponding to * in (A). Total Rab11a, Rab8a, and Rabin8 expression was normalized to GAPDH levels. (C–E) FRAP studies of ciliary tRFP-Rab8 in cells, treated with Dox for 24 h, with and without exogenous Rab11, Rabin8, and/or Rab14. Representative images of FRAP analysis performed on cilia (~5 μm) in cells treated with siControl or siGFP for 72 h, with the last 12 h serum starved to induce ciliation. Plots show the quantification of mobile fraction and half-life for the recovery of tRFP-Rab8a. (C) RPE tRFP-Rab8a+GFP-Rabin8 cells. (D) RPE tRFP-Rab8a+GFP-Rab11a cells. (E) RPE tRFP-Rab8a+GFP-Rabin8+miRFP670-Rab11a/miRFP670-Rab14 cells. Representative images from 10 cells analyzed and plots show mean ± SD from n = 3 experiments. **** p < 0.00001. Scale bar: 5 μm. See also .

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Western Blot, Control, Expressing

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A and B) Quantification of Rab8 effects on Rab11 ciliary localization. (A) Representative images of cilia in RPE (left) and RPE GFP-Rab8a (center) cells transiently transfected with tRFP-Rab WT and constitutively active (QL) proteins. (Right) Quantification of ciliary localization of tRFP-Rabs from cells starved for the last 12 h and stained with acetylated α-tubulin ( Ac Tub) antibody showing mean ± SEM from >80 cells from n = 3 experiments. (B) RPE tRFP-Rab8a+GFP-Rab11a cells were treated with siControl and sitRFP for 72 h and Dox for 24 h. (Left) Immunoblot probed with tRFP, Rab11a, and actin antibodies. (Center and right) Cells were serum starved the last 12 h and stained with Ac Tub antibodies. GFP-Rab11a ciliary localization was detected by spinning disk confocal microscopy (SDCM). (Center) Representative images of ciliated cells from a single xy-plane of a z stack. (Right) Quantification of ciliary GFP-Rab11a showing mean ± SEM for ~100 cells analyzed from n = 3 independent experiments. (C) Super-resolution imaging of Rab11 ciliary localization by Elyra 7 microscopy SIM 2 in fixed RPE tRFP-Rab8a+GFP-Rab11a cells, treated with Dox for 24 h, serum starved the last 12 h, and stained with CEP164 antibody. Images shown are from a single xy-plane from a z stack. The lower image shows the fluorescence line profile plots of tRFP-Rab8a and GFP-Rab11a corresponding to the dotted line. (D) Super-resolution time-lapse imaging of Rab11 and Rab8 localization during ciliogenesis. Cells described in (B) were Dox treated for 24 h, serum starved for 3 h ,and then imaged live using the Elyra 7 microscope SIM 2 every 10 min. Images shown are from a single xy-plane from a z stack. The lower image shows the fluorescence line profile plots as described in (C). Cilia are outlined in white. * p < 0.05; ** p < 0.001. Scale bar: 1 μm.

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Transfection, Staining, Western Blot, Confocal Microscopy, Imaging, Microscopy, Fluorescence

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A) Representative images of RPE tRFP-Rab8a+GFP-Rab11a, tRFP-Rab8a+GFP-Rabin8, and tRFP-Rab8a+GFP-Rabin8+miRFP670-Rab11a cells treated with Dox for 24 h and DMSO or CytoD for 30 min and imaged on an SDCM. Images shown are from a single xy-plane from a z stack. White arrows indicate colocalized vesicles or LTMs; green and magenta arrows indicate Rab11a and Rab8a vesicles that do not colocalize, respectively. (B) Quantification of tRFP-Rab8a LTMs in cells treated with siRNA for 72 h and Dox for the last 24 h, followed by DMSO or CytoD treatment for 30 min and imaged on SDCM. (Left) Representative single xy-plane image from a z stack of RPE tRFP-Rab8a treated with siRNA. (Right) Plot showing tRFP-Rab8a LTMs in cells treated with siRNA. Immunoblots (below immunofluorescence images) show protein expression from cell lysates stained with antibodies detecting actin and ablated proteins. Plot shows mean ± SEM for >100 cells from n = 3 independent experiments. (C) Characterization of Rabin8 dependence on Rab11-Rab8 colocalization on LTMs in zebrafish embryos. Immunoblot analysis (top left) of Rabin8 expression in 24 hpf MO-injected zebrafish embryos. Quantification of mNeonGreen-Rab8a+mCherry-Rab11a LTMs in embryos injected with mNeonGreen-Rab8a+mCherry-Rab11a with and without rabin8 MO (bottom left). Mean ± SEM for ~100 cells analyzed per condition from n = 5 and 6 independent experiments for uninjected and rabin8 MO, respectively. (Right) Representative images captured on an SDCM is shown. White arrows indicate colocalized LTMs; green and magenta arrows indicate Rab8a and Rab11a vesicles that do not colocalize, respectively. ns, not significant; * p < 0.05; ** p < 0.001; *** p < 0.0001. Scale bar: 5 μm. See also - , , and .

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Western Blot, Immunofluorescence, Expressing, Staining, Injection

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A) (Left) Quantification of tRFP-Rab8a LTMs in RPE tRFP-Rab8a+GFP-Rab11a cells treated with Dox for 24 h (mean ± SEM for ~100 cells from n = 3 experiments) imaged by Elyra 7 SIM 2 (images in ). (Right) tRFP-Rab8a colocalization with GFP-Rab11a on LTMs (calculated from Pearson’s correlation coefficient). Mean ± SEM from n = 3 experiments where Pearson’s correlation coefficient was calculated from 3 areas of each cell with 5 cells analyzed per experiment. (B) Representative images of RPE tRFP-Rab8a+GFP-Rabin8+miRFP670-Rab11a treated with Dox for 24 h followed by CytoD and imaged on Elyra 7 SIM 2 . tRFP-Rab8a+GFP-Rabin8 and tRFP-Rab8a+miRFP670-Rab11a colocalization on LTMs from the magnified regions of interest are shown below. Bottom images: (left plot) quantification of tRFP-Rab8a LTMs, (right plot) Pearson’s correlation coefficient for tRFP-Rab8a colocalization with GFP-Rabin8 or miRFP670-Rab11a on LTMs as described in (A). * p < 0.05; ** p < 0.001; *** p < 0.0001; **** p < 0.00001. Scale bar: 5 μm.

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques:

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A) (Right) Quantification of tRFP-positive LTMs in fixed RPE cells stably expressing tRFP-Rab8a and GFP-Rabin8 WT or GEF mutants (E192A and F201A) treated with Dox for 24 h, followed by CytoD for 30 min. (Left) Representative images from a single xy-plane of a z stack captured on an SDCM. Mean ± SEM for ~100 cells from n = 3 experiments. (B) Quantification of mNeonGreen-Rab8a and mCherry-Rab11a colocalized on LTMs in yolk sac cells from 24 hfp zebrafish embryos co-injected with Rabin8 or Rabin8 E192A mRNA. Mean ± SEM for n = 10 (Rabin8) and n = 9 (Rabin8 E192A) independent experiments. (C) Quantification of ciliation in cells described in (A) following serum starvation for 24 h and stained with Ac Tub. Mean ± SEM for ~100 cells from n = 3 experiments. (D) Quantification of FRAP recovery half-life and mobile fraction performed on cilia (~5 μm) in RPE tRFP-Rab8a+GFP-Rabin8 E192A and RPE tRFP-Rab8a+GFP-Rabin8 F201A cells treated with siControl or siGFP for 72 h with Dox treatment and serum starvation for the last 24 h and 12 h, respectively. Mean ± SD from n = 3 independent experiments. (E) Model of Rab11-Rab8 cascade membrane conversion meditated by Rabin8. * p < 0.05; ** p < 0.001; *** p < 0.0001; **** p < 0.00001. Scale bar: 5 μm. See also .

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Stable Transfection, Expressing, Injection, Staining, Membrane

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: (A) RPE cells transiently transfected with mCherry-Rab8a, GFP-Rabin8, and tBFP-Rab11b or BFP-Rab14 for 48 h. Representative images show a single xy-plane from a z stack captured on SDCM using a Hamamatsu Orca Flash4 camera. mCherry+tBFP colocalization is shown. (B) RPE GFP-Rabin8 cells transiently transfected with tBFP-Rab11b and mCherry-Rab8a, mCherry-Rab7 or mCherry-Rab6a for 48 h. Representative images show a single xy-plane from a z stack captured on SDCM with an EMCCD Evolve 512 camera. (C) RPE GFP-Rabin8+mCherry-Rab8a (Tet-inducible) cells transiently expressing tBFP-Rab11b and treated with Dox 24 h post-transfection imaged by time-lapse microscopy every 20 (representative images) or 25 min (plot). Representative images (top) show three-dimensional surface rendering of unfiltered z stack (step size, 1 μm) cell images for the time course. Image of BFP-Rab11b colocalization (white) with mCherry-Rab8a is shown at time point 450 min. Immunoblot shows expression of mCherry-Rab8a, detected with anti-Rab8 antibodies, and GFP-Rabin8, detected with GFP antibodies, following indicated Dox treatment times. Plot shows tBFP-Rab11b, mCherry-Rab8a, and GFP-Rabin8 fluorescence intensity in tBFP-positive vesicular structures (≥1 voxel) measured at the indicated times following Dox induction and plotted for five cells. Results shown as mean ± SD from two independent experiments. * p < 0.05; *** p < 0.0001. Scale bar: 5 μm.

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Transfection, Expressing, Time-lapse Microscopy, Western Blot, Fluorescence

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rab8a , Proteintech , Cat# 55296-1-AP; RRID: AB_10858398.

Techniques: Virus, Recombinant, Electron Microscopy, Sequencing, Software