Journal: Cell Communication and Signaling : CCS

Article Title: KLC3 regulates axonemal glutamylation via RAB11/FIP5 in polycystic kidney disease

doi: 10.1186/s12964-025-02475-7

Figure Lengend Snippet: KLC3 interacts with and regulates the RAB11/FIP5 complex. A , B Representative immunostaining images showing DAPI, KLC3, ( A ) RAB11FIP5, or ( B ) RAB11 in mIMCD-3 cells. C Co-IP of Flag-tagged empty vector (EV), KLC3, or KLC3ΔTPR from HEK293T cell lysates. The cells were transfected and were given the serum free media for 1 d. IP was performed using anti-FLAG. D , E For immunostaining, EGFP-tagged empty vector (EV), KLC3, or KLC3ΔTPR was transfected into mIMCD-3 cells. The transfected cells were given the serum free media for 3 d, and were stained with DAPI, acetylated-⍺-tubulin (Ac-⍺-tub), ( D ) RAB11FIP5, or ( E ) RAB11. Signal intensities at basal bodies of cilium were quantified using the ImageJ software. Data are presented as mean ± SD. Statistical significances were determined by one-way ANOVA. F - H Representative immunostaining images showing DAPI, Ac-⍺-tub, ( F ) RAB11FIP5, and ( G ) RAB11 in mIMCD-3 cells transfected with control or Klc3 siRNA under serum free condition. H Signal intensities at basal bodies of cilium were quantified using the ImageJ software. Data are presented as mean ± SD. Statistical significances were determined by unpaired two-tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Each triangle represents a value obtained from a single cell. All experiments were performed in at least three independent replicates

Article Snippet: The following primary antibodies were used: Phospho-AKT (Thr308) (4056), Phospho-AKT (Ser473) (4691), AKT (4691, CST); FLAG (F7425, Sigma); Polyglutamylation (AG-208-0020B; AdipoGen); Ac-alpha-tubulin (5335; Cell Signaling Technology); γ-tubulin (T6557, Sigma); RAB11FIP5 (ab153843; Abcam); RAB11FIP5 (14594-1-AP), ARL13B (17711-1-AP; Proteintech); RAB11 (700184, ThermoFisher); and β-actin (A300-491 A; Bethyl).

Techniques: Immunostaining, Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Staining, Software, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: KLC3 regulates axonemal glutamylation via RAB11/FIP5 in polycystic kidney disease

doi: 10.1186/s12964-025-02475-7

Figure Lengend Snippet: Expression of tubulin PTM and RAB11FIP5 in cyst-lining cells of ADPKD models. A , B Representative immunostaining images showing DAPI, Dolichos biflorus agglutinin (DBA, stained for collecting ducts), Ac-⍺-tub (K40), ( A ) GT335, and ( B ) RAB11FIP5 in Pkd1f/f and Pkd1f/f: HoxB7-cre kidneys at P13. C Graph shows Rab11FIP5 intensity of collecting duct cells in Pkd1f/f and Pkd1f/f: HoxB7-cre kidneys at P13. Data are presented as mean ± SD. Statistical significances were determined by unpaired two-tailed t -test. D Expression of Rab11Fip5 in whole kidney lysates from in Pkd1f/f and Pkd1f/f: HoxB7-cre kidneys at P13. Graphs show the ratios of Rab11Fip5 to β-actin, presented as mean ± SD. β-actin was used as a loading control. Statistical significances were determined by unpaired two-tailed t -test. E , F Representative immunostaining images showing DAPI, Ac-⍺-tub (K40), ( C ) GT335, and ( D ) RAB11FIP5 in non-ADPKD and ADPKD kidneys. G Representative images of in vitro cyst formation in mIMCD-3 cells transfected with the indicated siRNAs. Graph shows the cyst lumen size of indicated samples, presented as mean ± SD. Each circle represents a value obtained from a single cell. Statistical significances were determined by one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The following primary antibodies were used: Phospho-AKT (Thr308) (4056), Phospho-AKT (Ser473) (4691), AKT (4691, CST); FLAG (F7425, Sigma); Polyglutamylation (AG-208-0020B; AdipoGen); Ac-alpha-tubulin (5335; Cell Signaling Technology); γ-tubulin (T6557, Sigma); RAB11FIP5 (ab153843; Abcam); RAB11FIP5 (14594-1-AP), ARL13B (17711-1-AP; Proteintech); RAB11 (700184, ThermoFisher); and β-actin (A300-491 A; Bethyl).

Techniques: Expressing, Immunostaining, Staining, Two Tailed Test, Control, In Vitro, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: KLC3 regulates axonemal glutamylation via RAB11/FIP5 in polycystic kidney disease

doi: 10.1186/s12964-025-02475-7

Figure Lengend Snippet: Elongated axonemal glutamylation, accumulated ciliary IFT88, and cell proliferation decreased by depletion of KLC3 in ADPKD patient derived cyst lining cells. A , B Representative immunostaining images showing DAPI, Ac-⍺-tub, ( A ) GT335, and ( B ) IFT88 in human ADPKD patient derived cyst lining cells transfected with control or KLC3 siRNA under 1% serum starved condition. The scale bar indicates 2 μm. A Ac-⍺-tub length, GT335 length, ratio of GT335-to-Ac-⍺-tub, and ( B ) signal intensities of IFT88 protein at basal bodies and the distal end of each cilium were quantified using the ImageJ software. C CCK-8 assay in cells transfected with control or KLC3 siRNA for different days (0, 1, 2, 3 d). D The bar graph shows the relative mRNA levels of KLC3 in the siRNA-transfected cells. E , F Representative immunostaining images showing DAPI, Ac-⍺-tub, ( E ) GT335, and ( F ) IFT88 in human ADPKD patient derived cyst lining cells transfected with control or RAB11FIP5 siRNA under 1% serum starved condition. The scale bar indicates 2 μm. E Ac-⍺-tub length, GT335 length, ratio of GT335-to-Ac-⍺-tub, and ( F ) signal intensities of IFT88 protein at basal bodies and the distal end of each cilium were quantified using the ZEN software. G CCK-8 assay in cells transfected with control or RAB11FIP5 siRNA for different days (0, 1, 2, 3 d). H The bar graph shows the relative mRNA levels of RAB11FIP5 in the siRNA-transfected cells. All graphs are presented as mean ± SD. Statistical significances were determined by unpaired two-tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Each circle represents a value obtained from a single cell. All experiments were independently performed in triplicate

Article Snippet: The following primary antibodies were used: Phospho-AKT (Thr308) (4056), Phospho-AKT (Ser473) (4691), AKT (4691, CST); FLAG (F7425, Sigma); Polyglutamylation (AG-208-0020B; AdipoGen); Ac-alpha-tubulin (5335; Cell Signaling Technology); γ-tubulin (T6557, Sigma); RAB11FIP5 (ab153843; Abcam); RAB11FIP5 (14594-1-AP), ARL13B (17711-1-AP; Proteintech); RAB11 (700184, ThermoFisher); and β-actin (A300-491 A; Bethyl).

Techniques: Derivative Assay, Immunostaining, Transfection, Control, Software, CCK-8 Assay, Two Tailed Test

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 1 Extracellular vesicles derived from brain-seeking MDA-MB-231 cells (Br-EVs) modulate the expression of multiple Rab11 effector proteins (rab11fips) involved in intracytoplasmic vesicle trafficking and recycling. (a) Schematic representing degradation and recycling pathways in brain endothelial cells (BECs). Created using Biorender. (b) Schematic depicting the most widely acknowledged mechanisms of interaction between Rab11fip2, Rab11fip3, Rab11fip5, and the respective motor proteins for the tethering and transport of rab11 vesicles on cytoskeleton filaments. Created using Biorender. (c–e) Western blot quantification, representative immunofluorescent image, and fluorescence intensity quantification of Rab11fip2, (f–h) Rab11fip3, and (j–k) Rab11fip5 protein expression in BECs following treatment with PBS, P-EVs, and Br-EVs (mean ± SD; technical triplicates in 3

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Derivative Assay, Expressing, Western Blot, Fluorescence

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 2 Cerebral microvessels isolated from the brain cortex of mice treated with Br-EVs show decreased Rab11fip2 and increased Rab11fip3 and Rab11fip5 levels in response to Br-EV treatment. (a) Western blot quantification of Rab11fip2, Rab11fip3, and Rab11fip5 in cerebral microvessel lysates of mice treated with PBS, P-EVs, and Br-EVs. PBS, P-EV and Br-EV groups were run on separate gels under the same conditions and normalization to GAPDH was performed individually for each sample with raw values being presented. Uncropped gels are provided in the Figure S5 (mean ± SD; n = 4 for PBS and Br-EV groups and n = 6 and 5 for the P-EV group). (b) Representative fluorescent images of cerebral microvessels from the brains of mice treated with PBS, P-EVs, and Br-EVs and stained with Rab11fip2, Rab11fip3, and Rab11fip5 (green) and nuclei (blue). Scale bars 50 µm. (c) Quantification of the signal mean fluorescence intensity (MFI) of the cerebral microvessels of mice treated with PBS, P-EVs, and Br-EVs and stained for Rab11fip2, Rab11fip3, and Rab11fip5 (normalized to the image background; mean ± SD; n = 5 for the PBS group, n = 6 for the P-EV group, and n = 4 for the Br-EV group). Statistical analysis was performed using the Mann–Whitney test (a) and one-way ANOVA with Tukey’s multiple comparison test (c) (Wu and Voeltz 2021; Lee et al. 2019; Mohrmann et al. 2002; Busatto et al. 2020). ns = not significant; *p ≤0.0332; **p ≤0.0021; ****p ≤0.0001.

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Isolation, Western Blot, Staining, Fluorescence, MANN-WHITNEY, Comparison

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 5 BECs knocked down (KD) for Rab7 and overexpressing (OE) Rab11fip5 display motility and morphology patterns similar to those of BECs treated with Br-EVs. UMAP representation of BEC populations KD for Rab7 and Rab11fip2 and OE for Rab11fip3 and Rab11fip5 (from left to right), analysed for morphology features using (a) confocal microscopy (to be compared with Figure 3c) and (c) live cell imaging (to be compared with Figure 3j), and for motility features using (e) QPI (to be compared with Figure 4c) and (g) live cell imaging (to be compared with Figure 4j). (b, d, f and h) Tables showing variations in cluster distribution and the percentage of each cluster within each respective sample.

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Confocal Microscopy, Live Cell Imaging

Journal: bioRxiv

Article Title: FERARI: a novel tether involved in endocytic recycling

doi: 10.1101/500082

Figure Lengend Snippet: (A) CRISPR-Cas9 knock-outs of vipas39, vps45, rbsn and ank3 cause enlargement of Rab11 compartments in HeLa cells. Representative images from live cell imaging of Rab11-GFP in control and indicated knock-out cells. (B) Quantification of deconvolved and FIJI processed images shown in (A). n= 45-60 cells from two-three biologically independent experiments. Sizes are given in arbitrary units showing an enlargement of the compartments compared to mock treated cells. (C) Schematic representation of C. elegans intestinal cells and approximation of different compartments in the endosomal system (shown in white). (D) GFP-RAB-11 structures are changed by FERARI(RNAi) (D left side). Normal RAB-11 structures near the apical membrane (in wild-type) are greatly enlarged and mis-localized to more basal positions inside intestinal cells (indicated by arrowheads in D). RFP-RAB-10 compartments are also strongly affected by RNAi of FERARI components (D right side). The wild-type round compartments with small networks (see arrowhead) are changed to grossly enlarged networks in FERARI knock-down worms (arrowheads for spe-39, vps-45, rabs-5, rme-1 and unc-44 knock-downs). The enlarged globular compartments in spe-39(RNAi) worms (right panel) might be explained by an additional role of SPE-39 in the CHEVI complex. (E) Quantification of RAB-11 and RAB-10 phenotypes shown in (D). RAB-11 compartments and RAB-10 tubules were measured in 6 worms (10 structures each), RAB-10 globular compartments were quantified semi-automatically and comprise 50-120 structures (per worm) in 6 different worms.

Article Snippet: The antibodies used in this study: polyclonal rabbit anti-VIPAS39 (20771-1-AP; proteintech; 1:2000), polyclonal rabbit anti-Rab11-FIP5 (NBP1-81855; Novus Biologicals;1:2,000), polyclonal rabbit anti-EHD1 (NBP2-56035; Novus Biologicals; 1:2,000), polyclonal rabbit anti-rabenosyn-5 (NB300-813; Novus Biologicals; 1:2,000), monoclonal mouse anti-myc (9E10) (1:3,000 for WB and 1:200 for Immunostaining; Sigma-Aldrich; M4439), polyclonal rabbit anti-GFP (TP401; Torrey Pines; 1:3,000 for WB and 1:200 for Immunostaining).

Techniques: CRISPR, Live Cell Imaging, Knock-Out, Membrane

Journal: bioRxiv

Article Title: FERARI: a novel tether involved in endocytic recycling

doi: 10.1101/500082

Figure Lengend Snippet: (A) Rabenosyn-5 binds to Rab11FIP5 but not to Rab11FIP2 in HEK-293 cells. myc-tagged Rab11FIP5 but not Rab11FIP2 was co-immunoprecipitated with GFP-Rabenosyn-5. Representative images from 3 independent experiments. (B) Western blot of immunoprecipitation of endogenous Rab11FIP5 with GFP-Rab11 in HEK-293 cells. (C) Rab11 binds to FERARI through Rab11FIP5. HEK-293 cells were co-transfected with GFP-Rab11 and myc-VIPAS39 in the presence or absence of myc-Rab11FIP5. Interaction between VIPAS39 and Rab11 was enhanced in the presence of Rab11FIP5. (D) Live cell imaging of Rab11FIP5 CRISPR/Cas9 knock-out HeLa cells. Rab11 compartments were enlarged, similar to the phenotype of the knockout other FERARI subunits. (E) Quantification of deconvulated and FIJI processed images shown in (D). About 2,300 GFP-Rab11 positive particles were analyzed from 50 cells from two independent experiments. (F) Phenotypes of rfip-2 knock-downs in worm intestinal cells with GFP-RAB-11 (left) and RFP-RAB-10 (right) markers. Phenotypes are the same as for other FERARI components (see ), namely enlarged globular RAB-11 compartments and enhanced networks of RAB-10 (indicated by arrowheads). (G) Quantification of rfip-2 phenotypes shown in (F). (H) Interactions between proteins in the yeast two hybrid vectors were tested biochemically with pull-downs, because no growth was seen on plates lacking leucine. Worm RFIP-2 (Y39F10B.1) binds to RME-1 and SPE-39. Interaction between RME-1 and RABS-5 also occurs in worms.

Article Snippet: The antibodies used in this study: polyclonal rabbit anti-VIPAS39 (20771-1-AP; proteintech; 1:2000), polyclonal rabbit anti-Rab11-FIP5 (NBP1-81855; Novus Biologicals;1:2,000), polyclonal rabbit anti-EHD1 (NBP2-56035; Novus Biologicals; 1:2,000), polyclonal rabbit anti-rabenosyn-5 (NB300-813; Novus Biologicals; 1:2,000), monoclonal mouse anti-myc (9E10) (1:3,000 for WB and 1:200 for Immunostaining; Sigma-Aldrich; M4439), polyclonal rabbit anti-GFP (TP401; Torrey Pines; 1:3,000 for WB and 1:200 for Immunostaining).

Techniques: Immunoprecipitation, Western Blot, Transfection, Live Cell Imaging, CRISPR, Knock-Out

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: RAB11FIP5 Is Significantly Upregulated in Individuals Who Develop bnAbs, Related to Figure 1 (A) Heatmaps of metadata from the cohort of individuals studied. Natural log of geometric mean (ID50) neutralization and mean viral load from sampled time points in addition to sex and age. Age and sex did not differ significantly between the bnAb and control groups. A more detailed description of these subjects and attributes of the larger cohort from which they were selected are provided in Moody et al. (2016) . (B) Quantitative PCR for RAB11FIP5 expression from RNA isolated from individuals’ PBMCs. Cohort A bnAb n = 41; Cohort A control n = 25; Cohort B bnAb n = 21; Cohort B control n = 16. P value determined by Wilcoxon-Mann-Whitney. No statistically significant difference between the bnAb and Control group was detected for Cohort B samples alone. (C and D) Representative flow cytometry density plots demonstrating the populations sorted for quantitative PCR and RNA-seq. (E) RAB11FIP5 expression level measured by RNA-seq in immune subsets, the fraction of reads per million of mapped reads (FPM) graphed with SEM.

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Neutralization, Control, Real-time Polymerase Chain Reaction, Expressing, Isolation, MANN-WHITNEY, Flow Cytometry, RNA Sequencing

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: Upregulation of RAB11FIP5 in bnAb Individuals (A and B) Plots of differential transcript expression in the bnAb group compared with control group (A) and after controlling for age, sex, country, autoantibody status, and viral load (B). Transcripts with p < 0.05 and log (FC) >1 are colored in blue. Transcripts associated with vesicle trafficking are circled. (C) Boxplot of RAB11FIP5 expression levels for each individual in the bnAb (n = 47) and control group (n = 46; t test). (D and E) Spearman correlations of RAB11FIP5 expression (y axis) and neutralization breadth (principal component 1) (D) or viral load (E). bnAb group are in red and control group in blue; solid fill autoantibody positive and open fill autoantibody negative individuals. (F and G) Bar graphs of quantitative PCR of RAB11FIP5 of PBMC, CD19 + , CD4 + , CD8 + and non-B/T cells (F) and monocytes, NK, pDC and mDC cells (G). BnAb group (n = 3 or 4) shown in blue and control group (n = 3 or 4) shown in red. The groups of HIV-1 infected bnAb and control subjects selected for this analysis were matched for viral load. Group average and SEM shown. See also and and .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Expressing, Control, Neutralization, Real-time Polymerase Chain Reaction, Infection

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: More Pronounced NK Cell Subset Redistribution in bnAb versus Control Subjects (A) Representative pseudocolor flow cytometry plots from bnAb, control, and HIV-seronegative individuals showing NK cell subsets distinguished on the basis of CD56 and CD16 expression. Gated on live, CD19 − CD14 − CD4 − CD3 − lymphocytes. (B–D) Boxplots of the percentage of NK cells that were CD56bright (B), CD56dim (C), and CD56 − CD16 + (CD56neg) (D) in individuals in the bnAb (red; n = 22), control (blue; n = 19), and HIV-seronegative (black; n = 19) groups. Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles, and range. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. p values are corrected for multiple comparisons analysis and viral load. (E and F) Spearman correlations of (E) RAB11FIP5 mRNA levels in total PBMC and (F) plasma HIV-1 neutralization breadth (PC1) with the percentage of CD56dim (black) and CD56 − CD16 + (blue) NK subsets within total NK cells. See also Figure S3 and .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Control, Flow Cytometry, Expressing, Whisker Assay, Clinical Proteomics, Neutralization

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: Increased Frequencies of Mature and Adaptive-like NK Cells in bnAb Subjects (A–H) Boxplots of the percentage of total NK cells expressing NKG2A (A), NKG2C (B), iKIR (cocktail of antibodies against KIR2DL1/S5, KIR2DL2/L3/S2, KIR3DL2, KIR3DL1) (C), CD85j (D), CD57 (E), Siglec7 (F), FcεRI-γ (G), and PLZF (H) in bnAb (red; n = 22), control (blue; n = 19) and HIV seronegative individuals (black; n = 22). Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles, and range. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. p values are corrected for multiple comparisons analysis and viral load. (I) SPICE (simplified presentation of incredibly complex evaluations) analysis of CD57, FcεRI-γ, PLZF, and Siglec7 receptor combinations on total NK cells in HIV seronegative individuals (n = 22), control (n = 19) and bnAb (n = 22) subjects. SPICE pie charts are shown for seronegative, control and bnAb groups and for individuals with the lowest RAB11FIP5 mRNA (bottom 5; blue; n = 5) in the control group and highest levels of expression of RAB11FIP5 mRNA in total PBMC (top 5; red; n = 5) in the bnAb group. SPICE bar charts depict the proportion of total NK cells expressing the indicated receptor combinations in the top 5 RAB11FIP5 subjects in the bnAb group (red bars) and bottom 5 RAB11FIP5 subjects in the control group (blue bars). Student’s t test was used to compare samples in SPICE. + p < 0.0001. See also and .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Expressing, Control, Whisker Assay

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: RAB11FIP5 Transcript in Total PBMC Correlates with Receptor Expression on Total NK Cells, Related to Figure 3 (A–F) Spearman correlation of RAB11FIP5 mRNA levels in total PBMC with total NK cells expression of (A) NKG2A, (B) CD85j, (C) iKIR (cocktail of antibodies against KIR2DL1/S5, KIR2DL2/L3/S2, KIR3DL2, KIR3DL1), (D) Siglec 7, (E) FcεRI-γ, and (F) PLZF. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. P values corrected for MCA. (G) Plasma antibody binding to HCMV cell lysate antigen or recombinant HCMV gB antigen measured by ELISA and Bio-Rad clinical CMV assay for individuals in the bnAb (blue) and control group (red). Values displayed as log area under the curve. Cutoff for positivity for the IgG and IgM clinical assay was > 1.1.

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Expressing, Clinical Proteomics, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: Decreased Function of NK Cells in bnAb Subjects (A and B) Representative pseudocolor flow cytometry plots from individual seronegative, control, and bnAb groups (A) and summary boxplots for all individuals analyzed in each group (B) showing CD107a expression, IFN-γ production, and TNF-α production by total NK cells following stimulation with MHC class I low target cells. Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles, and range. ∗ p < 0.05, ∗∗ p < 0.01. p values are corrected for multiple comparisons analysis and viral load (bnAb, n = 20; control, n = 19; seronegative, n = 18 or 19). (C) Spearman correlation of RAB11FIP5 mRNA levels in total PBMC with percentage of total NK cells expressing CD107a, producing IFN-γ, and producing TNF-α in response to target cell stimulation. See also Figure S6 .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Flow Cytometry, Control, Expressing, Whisker Assay, Cell Stimulation

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: Transcript Expression from Bulk NK Cell RNA-Seq and Cytokine Secretion from Rab11Fip5-Expressing NK-92 Cells Measured by Luminex Assay Together with Analysis of Their Cytolytic Activity, Related to and (A) Median unique molecular identifiers (UMIs) and genes detected per cell in the scRNA-seq datasets. (B) Single-cell RNA-seq analysis was performed on CD56bright, dim and neg NK subsets isolated from a single donor by cell sorting. Violin plots of transcripts significantly upregulated in RAB11FIP5 -expressing cells (red) compared to cells not expressing RAB11FIP5 (blue). Data for each NK cell subset is shown separately for significant genes (determined by likelihood ratio test and p ≤ 0.05). Normalized transcript expression is shown on the y axis. (C) NK cells from three HIV-infected donors were sorted into subsets on the basis of CD56 expression and subjected to bulk RNA-seq. Reads were aligned to the human genome (Hg38) and Fragments Per Kilobases of transcript per Million mapped reads (FPKM) were determined for each donor in each subset for IFNG transcript expression. (D) NK-92/ RAB11FIP5 transduced cells and NK-92/empty vector control cells were stimulated with 500 ng/ml PMA and 5 μg/ml ionomycin for 2 hours. Supernatants were harvested and levels of GM-CSF, sCD137, IFN-γ, sFas, sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α and perforin were analyzed by Luminex assay. All the cytokines in range of the standard curves are shown. Error bars represent standard deviation of quadruplicate wells. Wilcoxon-Mann-Whitney was utilized for statistical analysis. ns, not significant; ∗∗∗ p ≤ 0.001. (E) Granzyme B (GzB)-based cytotoxicity assays performed using NK-92/ RAB11FIP5 (red) or NK-92/ZsGreen control (blue) cells as effectors and K562 cells as targets at different effector:target ratios. The percentage of cells positive for proteolytically active GzB is represented as % GzB activity. Data from 3 individual experiments, each of which was were performed in triplicate, are shown.

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Expressing, RNA Sequencing, Luminex, Activity Assay, Isolation, FACS, Infection, Plasmid Preparation, Control, Standard Deviation, MANN-WHITNEY

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: High RAB11FIP5 -Expressing Cells Are Enriched in the CD56 − CD16 + NK Cell Subset (A and B) Non-linear dimensionality reduction by t-distributed stochastic neighbor embedding and visualization of single-cell RNA-seq of sorted NK cell subsets (CD56bright, 2,891; CD56dim, 7,674; CD56negative, 11,677) showing (A) all cells and (B) RAB11FIP5 expressing cells. Each dot represents a single cell. (C) Dot plot of RAB11FIP5 expression in single cells in each NK cell subset. Normalized expression value of RAB11FIP5 shown on y axis. Proportion of cells with detectable RAB11FIP5 expression shown above. (D) Violin plots of transcripts significantly (likelihood ratio test; p < 0.05) upregulated in RAB11FIP5 -expressing cells (red) compared to cells not expressing RAB11FIP5 (blue). Normalized transcript expression is shown on the y axis. p values for each transcript are also shown. See also Figure S7 and .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Expressing, RNA Sequencing

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet: Overexpression of Rab11Fip5 in NK-92 Cells Increases Cytokine and Granzyme Release (A) Effect of Rab11Fip5 overexpression on IFN-γ production and degranulation (CD107a expression) of NK-92 cells in response to stimulation with PMA/ionomycin. NK-92/Rab11Fip5 (red symbols) and NK-92/ZsGreen cells (blue symbols) were stimulated with 500 ng/mL PMA and 5 μg/mL ionomycin for 2 hr in the presence of a CD107a antibody and the protein transport inhibitor monensin. Dots represent MFIs from 6 replicate wells. Significance determined by Wilcoxon-Mann-Whitney (ns, not significant; ∗∗ p < 0.01). (B) Granzyme B (GzB)-based cytotoxicity assays performed using NK-92/Rab11Fip5 (red) or NK-92/ZsGreen control (blue) cells as effectors and K562 cells as targets at different effector:target ratios. The percentage of cells positive for proteolytically active GzB is represented as % GzB activity. The average values from 3 independent experiments performed in triplicate are shown. (C) Western blot of Rab11Fip5 expression in NK-92 cells transduced with a RAB11FIP5 expression vector or zsGreen vector control, in the presence or absence of K562 target cell stimulation (5:1 effector:target ratio). Cells were analyzed at 10 hr post stimulation. GAPDH used as protein loading control. See also Figure S7 .

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Over Expression, Expressing, MANN-WHITNEY, Control, Activity Assay, Western Blot, Transduction, Plasmid Preparation, Cell Stimulation

Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet:

Article Snippet: Human Rab11FIP5 gene , Genscript , NCBI ACCESSION# NM_015470.2.

Techniques: Recombinant, Staining, Cell Isolation, Enzyme-linked Immunosorbent Assay, Marker, Library Quantification, Gene Expression, Sequencing, Software