Journal: Vaccines

Article Title: Control of Cytoskeletal Dynamics by β-Arrestin1/Myosin Vb Signaling Regulates Endosomal Sorting and Scavenging Activity of the Atypical Chemokine Receptor ACKR2

doi: 10.3390/vaccines8030542

Figure Lengend Snippet: Alteration of cytoskeletal dynamics causes ACKR2 missorting into recycling compartments. ( A , B ) Flow cytometry analysis of ACKR2 membrane expression in CHO-K1/ACKR2 cells transiently transfected with the indicated pEGFP-tagged plasmids after incubation (1 h) with vehicle or 1 µM latrunculin A (panel A ,) and 10 µM nocodazole (panel B ). Transfected but pEGFP-negative cells (pEGFP neg ) served as internal control of cells transfected and overexpressing dominant negative Rab4 (Rab4 S22N) and Rab11 (Rab11 S25N) pEGFP plasmids (pEGFP pos ). Results are representative of mean ± SEM of n = 3 experiments and are shown as percentage of MFI of pEGFP neg /pEGFP pos cells. ( C , D ) Confocal microscopy analysis of CHO-K1/ACKR2 cells incubated (1 h) with vehicle (DMSO), 1 µM latrunculin A (panel C ), or 10 µM nocodazole (panel D ). Left panels show ACKR2 staining (red) merged in panel C with Rab4 (blue) and actin (phalloidin staining in green), and in panel D with Rab11 (blue) and microtubules (α-tubulin staining in green). Panels on the right show Nomarski interference contrast merged with the colocalization channel. Quantification of ACKR2 colocalization with Rab4 or Rab11 is shown as the mean ± SEM of PCC evaluated in at least n = 20 cells. Data were analyzed by two-way ANOVA with Tukey’s post hoc test (panel A and B ), or unpaired Student’s t -test (panel C and D ). *: p ≤ 0.05, ***: p ≤ 0.005 vehicle versus inhibitor-treated cells, ###: p ≤ 0.005 pEGFP pos versus pEGFP neg cells.

Article Snippet: Lipofectamine 2000 and anti-human Rab11 antibody were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Expressing, Transfection, Incubation, Dominant Negative Mutation, Confocal Microscopy, Staining

Journal: Vaccines

Article Title: Control of Cytoskeletal Dynamics by β-Arrestin1/Myosin Vb Signaling Regulates Endosomal Sorting and Scavenging Activity of the Atypical Chemokine Receptor ACKR2

doi: 10.3390/vaccines8030542

Figure Lengend Snippet: Myosin Vb sustains ACKR2 upregulation and chemokine degradation. ( A , B ) Confocal microscopy analysis of CHO-K1/ACKR2 or CHO-K1/CCR5 cells stimulated with 100 nM CCL3L1 (1 h) and double stained for ACKR2 or CCR5 (red; panels A and B , respectively) and myosin Vb (green). Nuclear staining (DAPI) is in blue. Merged images are shown in left panels, right panels show colocalization channels, with quantification of ACKR2 or CCR5 colocalization with myosin Vb expressed as mean ± SEM of PCC evaluated in at least n = 30 cells. ( C , D ) Confocal microscopy analysis of CHO-K1/ACKR2 cells transiently transfected with a pEGFP-tagged myosin Vb tail and stimulated with 100 nM CCL3L1 (1 h) and double stained for ACKR2 (red) and microtubules (α-tubulin staining; green; panel C ) or Rab11 (green; panel D ). Nuclear staining (DAPI) is in blue. Left and right images refer to cells negative and positive for myosin Vb tail transfection, respectively. Graphs on the right report quantification of ACKR2 colocalization with microtubules (panel C ) and Rab11 (panel D ) shown as mean ± SEM of PCC evaluated in at least n = 15 cells (open bar: untreated cells, black bar: stimulated cells). ( E ) Flow cytometry analysis of ACKR2 constitutive internalization in transiently transfected pEGFP-negative CHO-K1/ACKR2 cells (open symbols) or expressing a pEGFP-tagged myosin Vb tail (closed symbols), evaluated as membrane expression after incubation with anti-ACKR2 antibody at 4 °C, and finally shifted to 37 °C for the indicated time, followed by incubation with a secondary antibody at 4 °C. ( F ) Flow cytometry analysis of ACKR2 membrane expression in unstimulated (open bars) or stimulated cells for 1 h with 100 nM of CCL3L1. ( G ) ACKR2 scavenging activity evaluated in sorted CHO-K1/ACKR2 cells not expressing (open symbols) or expressing a pEGFP-tagged myosin Vb tail (closed symbols) following incubation with 10 nM CCL3L1 at indicated time points. In panels E and F , results are shown as mean ± SEM of n = 3 experiments, whereas in panel G of n = 2 experiments. Data were analyzed by two-way ANOVA with Tukey’s post hoc test (panel C – F ), or unpaired Student’s t -test (panel A and B ). ***: p ≤ 0.005 stimulated versus untreated cells; ##: p ≤ 0.01, ###: p ≤ 0.005 negative versus positive, or untransfected versus myosin Vb tail cells.

Article Snippet: Lipofectamine 2000 and anti-human Rab11 antibody were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Confocal Microscopy, Staining, Transfection, Flow Cytometry, Expressing, Incubation, Activity Assay

Journal: Cell Death & Disease

Article Title: Inhibition of the miR-192/215–Rab11-FIP2 axis suppresses human gastric cancer progression

doi: 10.1038/s41419-018-0785-5

Figure Lengend Snippet: Expression levels of Rab11-FIP2 by IHC in GC, lymphatic metastatic, and adjacent normal tissues

Article Snippet: Human Rab11-FIP2 siRNA (siRNA1, siRNA2, and siRNA3 sequences were listed in the Supplementary Table ) were from Ribobio (Guangzhou, Ribobio, Co., Ltd).

Techniques: Expressing

Journal: Nature

Article Title: MYCT1 controls environmental sensing in human haematopoietic stem cells.

doi: 10.1038/s41586-024-07478-x

Figure Lengend Snippet: Fig. 4 | MYCT1 is located in endosomes and interacts with vesicle trafficking and receptor signalling machinery. a, The transmembrane topology of MYCT1 was predicted from its amino acid sequence using Phobius48 and is depicted as a schematic and histogram of probability. b,c, High-resolution Airyscan immunofluorescence images (b) and quantification (c) of the localization of overexpressed MYCT1–V5 in human CB HSPCs after 5 days of culture. MYCT1–V5 was visualized by staining for V5, and colocalization with endosomal markers (clathrin, RAB5, RAB7 and RAB11), Golgi marker (GM130) and mitochondrial marker HSP60 was evaluated. DAPI indicates nuclei. Colocalization channel (Coloc) shows areas positive for V5 and each marker. Scale bars, 3 µm (RAB5) or 2 µm (other columns). Analysis was performed using Imaris (v.9.7.2) software

Article Snippet: HSP60 D6F1 Cell Signaling Technology 12165T, reported recognize endogenous human HSP60 (manufacturer’s website) Lamin B1 EPR8985(B) Abcam ab133741, reported recognize human LaminB1 (manufacturer’s website) Na/K ATPase EP1845Y Abcam ab76020, reported recognize human Na/K ATPase (manufacturer’s website) phospho-AKT D9E Cell Signaling Technology 4060S, reported recognize endogenous human pAKT (Ser 473) (manufacturer’s website) phospho-ERK D13.14.4E Cell Signaling Technology 4370S, reported recognize endogenous human pERK1/2 (Thr202/Tyr204) (manufacturer’s website) Rab11 D4F5 Cell Signaling Technology 5589T, reported recognize endogenous human Rab11 (manufacturer’s website) Rab5 C8B1 Cell Signaling Technology 3547T, reported recognize endogenous human Rab5 (manufacturer’s website) Rab7 D95F2 Cell Signaling Technology 9367T, reported recognize endogenous human Rab7 (manufacturer’s website) V5 SV5-Pk1 ThermoFisher Scientific R960-25, reported recognize V5 tag (manufacturer’s website).

Techniques: Sequencing, Immunofluorescence, Staining, Marker, Software

Journal: Cells

Article Title: Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

doi: 10.3390/cells9051132

Figure Lengend Snippet: Kalirin regulates the activation of Rab11 and endocytic recycling of transferrin. ( A ) Kalirin immunoprecipitates containing GEF activities on Rab11. Triton solubilized cellular membranes from cells transfected with pEAK-His-myc-kalirin7 or empty vector were used for immunoprecipitation with anti-myc antibodies. Precipitated proteins were used for the [ 3 H]GDP release assay as described in Methods. Mean ± SD percentages of [ 3 H]GDP remaining on GST-Rab1-His, GST-Rab11-His, and GST-Rac1 were graphed (n = 3, two-tailed Student’s t -test: n/s, not significant, * p < 0.01). ( B ) Knockdown of kalirin reduced Rab11GEF activities in cellular membranes. After knocking down kalirin in NRK cells as in (2A), cellular membranes were prepared and extracted in GEF assay buffer containing triton X-100. Equal amounts of solubilized membranes from cells treated with eGFP-siRNA and kalirin-siRNA were used for [ 3 H]GDP release from GST-Rab1-His and GST-Rab11-His as above. BSA is a no-GEF control (n = 3, Mean ± SD, two-tailed Student’s t -test: n/s, not significant, * p < 0.01). ( C ) Synchronized uptake and recycling of Alexa568-transferrin was performed with siRNA-treated NRK cells for indicated times. After uptake, NRK cells on glass coverslips were processed for fluorescent microscopy. Shown are confocal images. Scale bar: 10 μm. Signal intensities of Alexa568-transferrin and the corresponding background of the images for each condition were measured using NIH ImageJ. After subtracting the background, the mean values of Alexa568-transferrin signals were calculated and plotted (n = 33 cells for kalirin-siRNA and 34 cells for eGFP-siRNA for 5 min incubation, n = 35 cells for kalirin-siRNA and 31 cells for eGFP-siRNA for 30min incubation; Mean±SD, two-tailed Student’s t -test: * p < 0.01).

Article Snippet: In brief, cells on glass coverslips were washed twice in PBS, fixed in 4% paraformaldehyde/PBS at room temperature for 15 min, quenched in 50 mM NH 4 Cl, washed twice in PBS and labeled with primary antibodies against pan -kalirin (polyclonal against the 4 to 7 spectrin repeats of kalirin; Millipore) and Rab11 (monoclonal against AA86-207 of human Rab11a; BD Biosciences).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Release Assay, Two Tailed Test, GEF Assay, Microscopy, Incubation

Journal: Cells

Article Title: Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

doi: 10.3390/cells9051132

Figure Lengend Snippet: Kalirin locates at Rab11 positive recycling endosomes. ( A ) Low frequency of co-localization between kalirin and Rab11 at steady state. NRK cells on glass coverslips were fixed and processed for labeling with antibodies for kalirin (green) and Rab11 (red). Shown are confocal images. Boxed regions are enlarged and shown underneath the corresponding image/channel. Arrowheads identify structures co-labeled with Rab11 and kalirin. Scale bar: 10 μm. ( B ) Enhanced co-localization of kalirin with Rab11 upon expression of Rab11S25N, a dominant negative mutant of Rab11. NRK cells on glass coverslips seeded in a 6-well plate were transfected with 0.3 μg of pcDNA 3 -Rab11S25N. After 5 hrs of culture, cells were processed for labeling Rab11 (green) and kalirin (red). Boxed regions are enlarged and shown underneath the corresponding image/channel. Arrows indicate structures containing both Rab11 and kalirin, whereas arrowheads identify Rab11 positive structures void of kalirin. The star symbol in each channel indicates a cell likely to be not transfected. Scale bar: 5 μm.

Article Snippet: In brief, cells on glass coverslips were washed twice in PBS, fixed in 4% paraformaldehyde/PBS at room temperature for 15 min, quenched in 50 mM NH 4 Cl, washed twice in PBS and labeled with primary antibodies against pan -kalirin (polyclonal against the 4 to 7 spectrin repeats of kalirin; Millipore) and Rab11 (monoclonal against AA86-207 of human Rab11a; BD Biosciences).

Techniques: Labeling, Expressing, Dominant Negative Mutation, Transfection

Journal: Cells

Article Title: Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

doi: 10.3390/cells9051132

Figure Lengend Snippet: Knockdown of kalirin induces cell shrinkage and tubulation of recycling endosomes. NRK cells were transfected with siRNAs for eGFP or kalirin as above and processed for labeling Rab11 (red), kalirin (green), and nuclei (blue). ( A ) Confocal images show that signals for kalirin were diffusely distributed and occasionally occurred at Rab11 positive structures in NRK cells treated with eGFP-specific siRNAs, whereas kalirin signals were concentrated at large punctate structures co-labeled with Rab11 in kalirin-siRNA treated NRK cells. Boxed areas are enlarged and shown beneath the corresponding images. Arrowheads point to structures containing both Rab11 and kalirin. Arrows in the red channel of kalirin-siRNA treated NRK cells identify elongated Rab11 positive recycling endosomes, which are rarely seen in eGFP-siRNA treated cells. Scale bars: 5 μm. ( B ) Comparison of cross-sectional areas of cells treated with eGFP-siRNA vs kalirin-siRNA. Each symbol in the plot represents one cell (Mean ± SD, Student’s t -test: #: p < 0.0001). ( C ) Comparison of Rab11 positive structures shows that there is a shift in size of Rab11 positive structures from smaller to larger ones in kalirin-siRNA treated cells relative to those in cells treated with eGFP-siRNA. Statistical significance was conducted with Chi square test for trend ( X 2 , df = 16.99, 1: p < 0.0001).

Article Snippet: In brief, cells on glass coverslips were washed twice in PBS, fixed in 4% paraformaldehyde/PBS at room temperature for 15 min, quenched in 50 mM NH 4 Cl, washed twice in PBS and labeled with primary antibodies against pan -kalirin (polyclonal against the 4 to 7 spectrin repeats of kalirin; Millipore) and Rab11 (monoclonal against AA86-207 of human Rab11a; BD Biosciences).

Techniques: Transfection, Labeling