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: Journal of Cancer

Article Title: Syntaxin 2 promotes colorectal cancer growth by increasing the secretion of exosomes

doi: 10.7150/jca.51494

Figure Lengend Snippet: The secretion of exosomes in CRC cells could be modulated by STX2. (A, B) Electron microscopy images of exosomes purified from cell culture supernatants. Scale bar, 50 nm. (C, D) Equal amounts of total exosomal proteins (quantified by Bradford assay) secreted by control over-expressed or knockdown cells were analyzed by Western blot for the presence of CD63, CD81 and TSG 101 proteins. (E) Size distribution of SW480-secreted vesicles as analyzed by NTA. (F) Size distribution of LOVO-secreted vesicles as analyzed by NTA. (G) Co-IP analysis of the interaction between STX2 and Rab8a in SW480. (H) Western blot analysis of CD63, CD81 and TSG 101 proteins in equal amounts of total exosomal proteins (quantified by Bradford assay) secreted by SW480 cells with different expression of STX2 and Rab8a.

Article Snippet: 5% non-fat dry milk was used to block the membranes and then the membranes were incubated overnight at 4°C with the primary antibodies of STX2 (Proteintech, USA), Rab8a (Proteintech, USA), CD63 (Proteintech, USA), CD81 (Santa Cruz Biotechnology, USA), TSG101(Santa Cruz Biotechnology, USA) and α-Tubulin (Sigma, USA).

Techniques: Electron Microscopy, Purification, Cell Culture, Bradford Assay, Control, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Expressing

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