Journal: bioRxiv

Article Title: N 6 -methyladenosine binding proteins negatively regulate HIV-1 infectivity and viral production

doi: 10.1101/257410

Figure Lengend Snippet: (A) Surface levels of CD4 (exogenous) and CXCR4 (endogenous) in HeLa/CD4 cells overexpressing individual Y1-3 proteins or vector control cells were analyzed by flow cytometry. Isotype-matched IgG was used as a negative control for immunostaining. The numbers on the top of the plots indicate the percentages of CD4-and CXCR4-positive cells. (B-F) HeLa/CD4 cells overexpressing individual Y1-3 proteins were infected with HIV-1 NL4-3 (MOI=1) for 72 h. (B) HIV-1 Gag protein expression and over-expression of FLAG-tagged Y1-3 proteins in HeLa/CD4 cells were confirmed by immunoblotting. (C) ELISA quantification of HIV-1 p24 levels in the supernatants from infected cells. (D and E) qPCR quantification of the levels of HIV-1 early RT products (D) and late RT products (E). (F) RT-qPCR quantification of HIV-1 gag mRNA in the cells. (C-F) The reverse transcriptase inhibitor nevirapine (NVP) was used to treat vector cells as a negative control for HIV-1 infection. All results are shown as mean ± SEM (n=3) and data presented are representative of 3 independent experiments. Dunnett’s multiple comparison test was used to determine statistical significance. ** P <0.005 and *** P <0.0005 compared with vector control cells.

Article Snippet: HeLa/CD4 cells were treated with the non-enzymatic cell dissociation solution (C5914, Sigma-Aldrich) and double stained with FITC-conjugated CD4 (MHCCD0401-4, Thermo Fisher) and phycoerythrin-conjugated CXCR4 (555974, BD Pharmingen) antibodies as described [ ].

Techniques: Plasmid Preparation, Flow Cytometry, Negative Control, Immunostaining, Infection, Expressing, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: Confocal analysis of membrane lipid rafts in purified murine SKL cells . Panel A. Defective lipid raft formation in murine C57Bl/6 Nlrp3-KO BM-purified SKL cells or wild type BM-purified SKL cells exposed to adenosine (10 μM) or CoPP (50 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml), stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control) but not in SKL cells isolated from Nlrp3-KO animals or SKL cells isolated from WT animals after adenosine and CoPP treatment. Panel B. The chemotactic responsiveness of mBMMNCs untreated or treated with 10 Panx, SC Panx, or apyrase in unsupplemented medium or medium supplemented with SDF-1, S1P, or ATP, as determined by counting the number of CFU-GM clonogenic progenitors. Results are combined from two independent experiments. *p > 0.05, ** p > 0.01, *** p > 0.001

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Membrane, Purification, Staining, Marker, Confocal Microscopy, Control, Isolation

Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: The impact of pannexin 1 channel blockade on short- and long-term engraftment of HSPCs in WT mice. Panel A. Lethally irradiated WT mice (9 per group) were transplanted with bone marrow mononuclear cells (BMMNCs) that had been previously labeled with a PKH67 cell linker and then treated with 10 Panx or SC Panx. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67 + cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx or SC Panx, and 12 days after transplantation the femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed for counting the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). * p < 0.05, ** p < 0.01, *** p < 0.001. Panel C. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx. White blood cells (above) and platelets (below) were counted at intervals (at 0, 3, 7, 14, 21, and 28 days after transplantation). * p < 0.05. Panel D . Defective lipid raft formation in murine BM-purified SKL cells from C57Bl/6 mice exposed to 10 Panx inhibitory peptide (200 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml) (positive control) or exposed to 10 Panx inhibitory peptide, stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control), but not in SKL cells after 10 Panx inhibitory peptide treatment

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Irradiation, Labeling, Transplantation Assay, In Vitro, Colony Assay, Control, Purification, Positive Control, Staining, Marker, Confocal Microscopy, Membrane

Journal: Cell Reports

Article Title: Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity

doi: 10.1016/j.celrep.2018.07.033

Figure Lengend Snippet: Flow Cytometric and scRNAseq Analyses Confirm the Identity of the pDCs and cDC1s Generated from Human CD34 + CB Cells and Identify Heterogeneity within cDC1s (A) viSNE plots from live Lin − HLA-DR + cells harvested from 18 day OP9+OP9_DLL1 FT7 cultures. Color codes indicate relative levels of marker cell-surface expression. (B) Overlay of the viSNE plot shown in (A) with the manually gated populations indicated in the legend. pDCs were gated as BDCA2 + CD123 + and cDC1s as BDCA2 − CD123 neg-to-low CADM1 + CD1c + . In the remaining CADM1 − cells, CD1c + cells were subdivided into BTLA − CX3CR1 + and BTLA + cells. Plots are representative of 4 donors from 2 experiments. (C) Lineage − HLA-DR + single cells were index sorted for phenotypes of cDC1s (CD141 + CADM1 + ), pDCs (CADM1 − CD123 + BDCA2 + ), putative pre-cDC2s (CADM1 − BDCA2 − CD1c + BTLA + ), or putative cDC2s (CADM1 − BDCA2 − CD1c + BTLA − ), as indicated by the symbols in the graphical legend. scRNAseq was performed using the SmartSeq2 protocol, followed by unsupervised dimensional reduction of the data using t-distributed stochastic neighbor embedding (t-SNE) with graph-based clustering. 7 cell clusters were identified as indicated by the color code in the graphical legend. (D) Violin plots showing mRNA expression profiles of previously known MNP-type-specific genes across all individual cells and in comparison between clusters identified in (A). (E) Enrichment analyses performed on each cell using connectivity map (cMAP), with independently generated and previously published human MNP-type-specific transcriptomic fingerprints. Results are presented as violin plots showing cell densities for the different cMAP scores (y axis) across the cell clusters (x axis) identified in (A). cMAP scores close to 1 versus −1 indicate strong enrichment of the signature of the cell type labeled on top versus bottom of the y axis. (F) Violin plots showing expression profiles of differentially expressed genes between clusters 6 and 7 (cDC1s). Individual dots represent single cells (C, D, and F). Data in (C)–(F) represent one experiment using one donor. (G) Protein expression of CLEC9A and CXCR4 on Lin − HLA-DR + CADM1 + BDCA2 − XCR1 − and XCR1 + cDC1s from FT7 cultures analyzed by flow cytometry. Plots are representative of 3 experiments and 5 donors. (H) Protein expression of CLEC9A and XCR1 on Lin − HLA-DR + CD11c + CD1c − CADM1 + cDC1s from adult peripheral blood analyzed by flow cytometry. Plots are representative of 3 experiments encompassing 6 different donors. (I) XCR1 protein expression on adult peripheral blood cDC1s sorted as Lin − HLA-DR + CD11c + CD1c − CD141 + CADM1 + CLEC9A + and either XCR1 + or XCR1 − 14 days after in vitro culture on OP9-DLL1 cells with the FT7+G cytokine cocktail. One experiment representative of three is shown. Dotted line, fluorescent minus one control; plain line, XCR1 staining. (J) Expansion of the sorted XCR1 − subset of adult peripheral blood cDC1s after 14 days of in vitro culture on OP9-DLL1 cells with the FT7 cytokine cocktail supplemented with GM-CSF (1 ng/mL). Data shown are from 3 experiments, each with a different donor (symbols), with mean ± Sd shown across the three donors for each experimental condition. See also and .

Article Snippet: Mouse Anti-Human CXCR4, APC conjugate, Clone 12G5, dilution 1:50 , Miltenyi Biotec , Cat # 130-098-357.

Techniques: Generated, Marker, Expressing, Comparison, Labeling, Flow Cytometry, In Vitro, Control, Staining

Journal: Cell Reports

Article Title: Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity

doi: 10.1016/j.celrep.2018.07.033

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human CXCR4, APC conjugate, Clone 12G5, dilution 1:50 , Miltenyi Biotec , Cat # 130-098-357.

Techniques: Purification, Recombinant, Binding Assay, Staining, Antibody Labeling, Reverse Transcription, Multiplex Assay, In Vitro, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Secondary damage and neuroinflammation in the spinal dorsal horn mediate post-thalamic hemorrhagic stroke pain hypersensitivity: SDF1-CXCR4 signaling mediation

doi: 10.3389/fnmol.2022.911476

Figure Lengend Snippet: Secondary neuroinflammation in the spinal dorsal horn 7 days after ITS or ITC. (A) Representative immunofluorescent photomicrographs showing the expression of Iba-1, GFAP, SDF1, and CXCR4 at the spinal dorsal horn contralateral to ITS or ITC. (B) Quantification of the mean immunofluorescent intensity of Iba-1, GFAP, SDF1, and CXCR4-labeled cellular profiles. *** p < 0.001 ITC vs. ITS, n = 3 rats/group. (C) Representative immunofluorescent photomicrographs showing co-localization of CXCR4 with NeuN, Iba-1, and GFAP-labeled cellular profiles at the spinal dorsal horn contralateral to ITC by orthogonal viewing method. (D) The percentage of CXCR4 in NeuN, Iba-1, and GFAP-labeled cellular profiles. (E) Western blot assay of Iba-1, GFAP, SDF1, and CXCR4 in bilateral dorsal spinal cord 7 days after ITS or ITC. (F) Quantitative analysis of the Western blot results for Iba-1, GFAP, SDF1, and CXCR4 in bilateral dorsal cord following 7 days after ITS or ITC. * p < 0.05, ** p < 0.01, ITC vs. ITS, n = 5 rats/group.

Article Snippet: After blocking with 2% BSA in 0.1 M PBS, the sections were incubated overnight at 4°C with the primary antibodies: mouse anti-NeuN (1:200, Abcam), rabbit anti-Bax (1:200, Abcam), rabbit anti-Bcl-2 (1:200, Abcam), goat anti-Iba1(1:200, Abcam), mouse anti-GFAP(1:200, Millipore), mouse anti-SDF1 (1:200, Santa), and rabbit anti-CXCR4 (1:400, Alomone) (for details refer to ).

Techniques: Expressing, Labeling, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Secondary damage and neuroinflammation in the spinal dorsal horn mediate post-thalamic hemorrhagic stroke pain hypersensitivity: SDF1-CXCR4 signaling mediation

doi: 10.3389/fnmol.2022.911476

Figure Lengend Snippet: Suppressing effects of i.t. antineuroinflammation treatment on expression levels of Iba-1, GFAP, SDF1, and CXCR4 in the spinal dorsal horn following ITS or ITC. Western blot assays (A) and quantitative analysis (B–C) of Iba1, GFAP, SDF-1, and CXCR4 expressions in the dorsal cord following i.t. administration of vehicle and drugs 10 days after ITS or ITC (3 days after i.t. catheterization). ITC-Veh, rats with ITC receiving i.t. administration of vehicle; ITC-MC, rats with ITC receiving i.t. administration of MC; ITC-FC, rats with ITC receiving i.t. administration of FC; ITC-AMD, rats with ITC receiving i.t. administration of AMD3100. * p < 0.05, ** p < 0.01, ITS vs. ITC-Veh; # p < 0.05, ## p < 0.01, ### p < 0.001, ITC-MC/ITC-FC/ITC-AMD vs. ITC-Veh; n = 5 rats/group.

Article Snippet: After blocking with 2% BSA in 0.1 M PBS, the sections were incubated overnight at 4°C with the primary antibodies: mouse anti-NeuN (1:200, Abcam), rabbit anti-Bax (1:200, Abcam), rabbit anti-Bcl-2 (1:200, Abcam), goat anti-Iba1(1:200, Abcam), mouse anti-GFAP(1:200, Millipore), mouse anti-SDF1 (1:200, Santa), and rabbit anti-CXCR4 (1:400, Alomone) (for details refer to ).

Techniques: Expressing, Western Blot

Journal:

Article Title: Adoptive immunotherapy of prostate cancer bone lesions using redirected effector lymphocytes

doi: 10.1172/JCI200422284

Figure Lengend Snippet: Migration of T bodies to the BM of preconditioned mice is mediated in part by CXCR4. SCID mice (n = 3 mice per group) were irradiated with 2 Gy TBI or were injected with 200 mg/kg cyclophosphamide, 24 hours before i.v. administration of erbB2-specific CR–bearing lymphocytes. T bodies were preincubated for 30 minutes on ice with mAb against CXCR4 (αCXCR4), irrelevant control mAb against human TfR (αTfR), or no antibody (none) and then were injected systemically into the mice. After 24 hours, BM was extracted and the number of human lymphocytes was measured by FACS analysis (detecting GFP-positive T bodies in the irradiation experiment and CFSE-labeled cells in the cyclophosphamide experiment). Treatment with anti-CXCR4 was significantly different from control treatment (anti-TfR) for both irradiated mice (*P = 0.003) and cyclophosphamide-treated mice (**P = 0.009) and was significantly different from no antibody treatment (P = 0.01 and P = 0.0002, respectively).

Article Snippet: The 12G5 mAb against human CXCR4 (R&D Systems Inc.) was used as indicated in Figures and .

Techniques: Migration, Irradiation, Injection, Control, Labeling

Journal:

Article Title: Adoptive immunotherapy of prostate cancer bone lesions using redirected effector lymphocytes

doi: 10.1172/JCI200422284

Figure Lengend Snippet: Transwell migration of T bodies toward recombinant human SDF-1α (rhSDF-1α) is dose dependent and is inhibited by anti-CXCR4. (A and B) Transwell migration of T bodies. (A) T bodies migrate toward increasing concentrations of rhSDF-1α. (B) T bodies were preincubated for 30 minutes on ice with anti-CXCR4 or anti-TfR prior to migration for 1 hour toward medium containing rhSDF-1α (150 ng/ml) or medium alone. Anti-CXCR4 significantly inhibited migration compared with anti-TfR or no treatment (*P = 0.016 and *P = 0.019, respectively).

Article Snippet: The 12G5 mAb against human CXCR4 (R&D Systems Inc.) was used as indicated in Figures and .

Techniques: Migration, Recombinant

Journal: PLOS Pathogens

Article Title: Alternative polyadenylation upon CPSF6 knock-out enhances HIV-1 infection in primary T cells

doi: 10.1371/journal.ppat.1013745

Figure Lengend Snippet: a, Schematic shows experimental workflow for RNA-Seq. Primary CD4+ T cells were isolated from the blood of 3 independent donors and edited via electroporation of 2 crRNPs targeting CPSF6 and TRIM5α alongside NT controls. RNA was extracted at day 5 post-electroporation and used for bulk RNA-Seq. In parallel, additional cells were used for viability assays, protein knock-out verification, and spreading infection assays. b, Immunoblot shows knock-out of CPSF6 and TRIM5α in primary CD4+ T cell protein lysates harvested at day 5 post-editing in one representative biological replicate (donor E). c, HIV-1 infectivity (% GFP positive cells normalized to NT control per donor) at day 5 post-challenge with HIV-1 NL4-3 nef:IRES:GFP in indicated knock-out primary CD4+ T cells from 3 biological replicates (donors E-G) as assessed by flow cytometry. Each bar represents the average of technical triplicates + /- SD with individual data points shown. Statistics were calculated relative to the NT control per condition by one-way ANOVA with Dunnet’s test for multiple comparisons; ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. d, Volcano plot shows differentially expressed genes in RNA-Seq data from uninfected CPSF6 knock-out primary CD4+ T cells as compared to NT controls in 3 biological replicates (donors E-G). Dots show significantly downregulated (blue) and upregulated (red) genes, dotted line shows threshold of 0.05 for adjusted p-value. e, Volcano plot shows differentially expressed genes in RNA-Seq data from uninfected TRIM5α knock-out primary CD4+ T cells as compared to NT controls in 3 biological replicates (donors E-G). Dots show significantly downregulated (blue) and upregulated (red) genes, dotted line shows threshold of 0.05 for adjusted p-value. f, Charts show functional enrichment (Metascape) analysis of top 20 downregulated (left, blue) and upregulated (right, red) pathways in differentially expressed genes from CPSF6 knock-out primary CD4+ T cells as compared to NT controls in 3 biological replicates (donors E-G). g, Histograms show fluorescence intensity of CD4 (first), CXCR4 (second), CCR5 (third), and CD25 (fourth) in primary CD4+ T cells from 1 representative biological replicate (donor I) at day 5 post-editing as measured by immunostaining and flow cytometry. Y-axis is normalized to the mode, histogram visualized using FlowJo version 10.10.

Article Snippet: 1x10 5 cells per condition were used for cell surface marker staining for CXCR4 (APC, Miltenyi Biotec, #130-120-708 [clone REA649]), CD4 (PE, Miltenyi Biotec, #130-113-225 [clone REA623]), CD25 (APC, Miltenyi Biotec, #130-115-535 [clone REA945]), and CCR5 (APC, Miltenyi Biotec, #130-120-708 [clone REA245]) at day 5 post-editing.

Techniques: RNA Sequencing, Isolation, Electroporation, Knock-Out, Infection, Western Blot, Control, Flow Cytometry, Functional Assay, Fluorescence, Immunostaining