Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: Changes in peripheral blood monocyte subsets in CTEPH patients. (A) UMAP plot of Mononuclear Phagocyte System (MPS) was divided into three cell types: monocytes, macrophages, and conventional dendritic cells (cDCs). Subsequently, monocytes were further divided into CD14+ monocytes and CD16+ monocytes. (B) A dot plot was used to annotate marker genes for CD14+ monocytes and CD16+ monocytes, along with a heatmap displaying the top 10 differentially expressed genes between the two cell types. (C) Ro/e (ratio of observed cell number to expected cell number) revealed the proportion of CD16+ monocytes in the CTEPH-N, CTEPH-I and HC groups. (D) The heat map showed the degree of correlation, and the numbers in the graph represented the correlation coefficient. (6MWD: 6-minute walk distance; PVR: pulmonary vascular resistance) (E) Schematic diagram of the gating strategy for distinguishing CD14+ monocytes from CD16+ monocytes by flow cytometry. (F) Differences in the proportion of CD16+ monocyte subsets in total monocytes between CTEPD patients ( n = 15) and matched healthy controls ( n = 15) were assessed by flow cytometry. All data were presented as means ± SEM, *** P < 0.001.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Marker, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: The functional characteristics of peripheral blood CD16+ monocytes in CTEPH patients. (A) GO analysis (biological process) of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (B) KEGG analysis of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (C) GSEA bar plot of CD16+ monocytes versus CD14+ monocytes in patients with CTEPH. (D) GO analysis (biological process) of upregulated genes in CD16+ monocytes between the CTEPH and healthy control samples. (E) KEGG analysis of upregulated genes in CD16+ monocytes between CTEPH patients and healthy controls. (F) GSVA heatmap of CD16+ monocytes between CTEPH patients and healthy controls. (G) Heat map of transcription factors upregulated in CD16+ monocytes between CTEPH patients and healthy controls.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Functional Assay, Control

Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: Characteristics analysis of CD16+ monocytes differentiation into IL-1β+ macrophages in peripheral blood of CTEPH patients. (A) Heatmap shows the correlation of CD16+ monocytes with four macrophage subgroups. (B) Resident macrophage scores of four macrophage subgroups. (C) Immunofluorescence images of CD68 (red), IL-1β (green) and DAPI (blue) in inferior vena cava thrombus tissue at different time points (1 day, 7 days and 14 days after ligation). Scale bar = 300 µm. (D) Pseudotime analysis of the developmental trajectory of CD16+ monocytes and Macrophages 2. (E) The developmental trajectory of CD16+ monocytes and Macrophages 2 was clustered into three clusters. (F) Genes with significant expression changes along the pseudotime trajectory. (G) KEGG pathway enrichment analysis along the pseudotime of CD16+ monocytes differentiation into Macrophages 2.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Immunofluorescence, Ligation, Expressing

Journal: bioRxiv

Article Title: High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

doi: 10.1101/2023.03.23.533972

Figure Lengend Snippet: HH exposure causes a decrease in the number of macrophages in spleen. The C57BL/6 mice were treated with NN and HH for 7 or 14 days, and the spleen and blood were collected for subsequent detection. ( A ) Visualization of spleen cell clusters using uniform manifold approximation and projection (UMAP). Each color represents a cluster of specific cells of the spleen, which was identified by a defined gene expression profile. ( B ) The proportions of different macrophage clusters derived from either NN-or HH-treated mouse spleens were analysed. ( C ) Individual macrophages derived from either NN-or HH-treated mouse spleens are denoted. ( D ) Calcein/PI double staining was analysed by flow cytometry. ( E and F ) Cell death proportions in total splenic cells are given as bar graphs. ( G and J ) Western blot detecting CD16 and CD206 protein expression in the spleen. ( H-I and K-L ) Statistical analysis of CD16 and CD206 expression in H and K. ( M ) F4/80 staining with CD11b, CD86 or CD206 in splenic cells was analysed by flow cytometry. ( N ) qPCR analysis of CCL2 and CCL7 expression in the spleen. ( O ) qPCR analysis of Csf1 and Csf2 expression in the spleen. ( P ) Flow cytometry detection of CD111b and Ly6C double-stained cells in the BM and spleen. CD11b hi Ly6C hi cell proportions in the BM ( Q-R ) and spleen ( S-T ) are given as bar graphs. ( U ) HO-1 and F4/80 expression in the spleen after HH exposure for 7 and 14 days. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

Article Snippet: The membranes were incubated with the following antibodies: HO-1 (1:2000, Abcam, ab13243, USA); Ft-L (1:1000, Abcam, ab69090, USA); Ft-H (1:1000, Novus, NBP1-31944, USA); NCOA4 (1:1000, Santa Cruz, sc-373739, USA); TfR (1:1000, Thermo Fisher, 13-6800, USA); Fpn (1:1000, Novus, NBP1-21502, USA); ACSL4 (1:1000, Santa Cruz, sc-271800, USA); xCT (1:1000, Proteintech, 26864-1-AP, USA); Gpx4 (1:1000, Abcam, ab125066, USA); CD206 (1:1000, RD, AF2535, USA); CD16 (1:2000, RD, AF1960, USA); and β-actin (1:1000, Sigma‒Aldrich, A5316, USA).

Techniques: Expressing, Derivative Assay, Double Staining, Flow Cytometry, Western Blot, Staining

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 2 Clustered, activated CD16/7-nephrinCD and CD16/7-Neph1CD induce lamellipo- dia formation in cultured human podocytes. (A) CD16/7-nephrinCD or CD16/7- Neph1CD was coexpressed with actin-GFP in human podocytes, which were activated by clustering as in Figure 1. Transfected cells with structures typical of lamellipodia on more than 30% of their circumferences were counted as cells with lamellipodial protrusions. (B) Using the same system as in A, cells were treated with PP2 or PP3 before activation. Transfected cells with actin tails longer than 5 μm or lamellipodia extending over more than 30% of the cell circumfer- ence were counted positive for these features. 20 transfected cells total were counted per experiment. Data (mean ± SEM) are from single experiments and are representative of 3 unique experiments.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Cell Culture, Transfection, Activation Assay

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 3 Crk1/2 knockdown attenuates nephrin-induced lamellipodial protrusive activity. (A) Immunoblot demonstrating attenuation of Crk1/2 expres- sion in human podocyte cell lines stably expressing 1 of 5 different shRNA constructs targeting the Crk1/2 gene. GAPDH protein expres- sion served as loading control. (B) Podocytes stably expressing the indicated Crk1/2 shRNA constructs were transfected with CD16/7- nephrinCD and actin-GFP and were activated by clustering as in Figure 1. The fraction of cells exhibiting lamellipodial protrusions was evaluated after fixation. (C) Immunoblot showing that Crk2-GFP or mouse Crk1 were reexpressed in stably knocked down human Crk shRNA3 podocytes. Mm, Mus musculus. (D) Crk shRNA3 podocytes were transfected with CD16/7-nephrinCD and actin-GFP and activated by clustering. After fixation, the fraction of cells exhibiting lamellipodial protrusions was determined. Results (mean ± SEM) are representative of 3 independent experiments.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Knockdown, Activity Assay, Western Blot, Stable Transfection, Expressing, shRNA, Construct, Control, Transfection

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 4 Nephrin-Crk association and nephrin-induced lamellipodial protrusions rely on nephrin tyrosine residues. (A) CD16/7-nephrinCD plasmids expressed in cultured podocytes were activated by clustering as in Figure 1 and analyzed by confocal microscopy. Podocytes pretreated with LY294002 before clustering are also shown. Crk was stained with anti-myc antibody and Alexa Fluor 488–conjugated secondary antibody. Origi- nal magnification, ×630. yz plane reconstructions are shown at far right. (B) Podocytes expressing CD16/7-nephrinCD or the indicated mutants and actin-GFP were activated with or without PP2, PP3, or LY294002, and the fraction of cells with lamellipodia was determined. Cells treated without primary CD16 antibody served as controls. Data (mean ± SEM) are representative of 3 experiments.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Cell Culture, Confocal Microscopy, Staining, Expressing

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 5 Nephrin engagement leads to phosphorylation of Cas, induces Cas-Crk complex formation, and results in Cas-Crk interaction– dependent lamellipodia formation. (A) Coimmunoprecipitation and immunoblots using indicated antibodies demonstrated tyrosine phosphorylation of Cas and increased Cas-Crk complex formation after CD16/7-nephrinCD clustering in cultured podocytes. (B and C) Cas-null, Cas-WT, and Cas-15F MEFs were stably transduced to express CD16/7-nephrinCD or the indicated mutants. Cells were activated by clustering as in Figure 1. Actin was stained with Alexa Fluor 488–labeled phalloidin. (B) Cells imaged by fluores- cence microscopy. Original magnification, ×630. (C) Lamellipodia per cell were counted. Data (mean ± SEM) are representative of 3 independent experiments.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Phospho-proteomics, Western Blot, Cell Culture, Stable Transfection, Staining, Labeling, Microscopy

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 6 Cas is recruited to clustered nephrin in a PI3K-dependent fashion, while its phosphorylation depends on Y5,7,10. (A) Nephrin coimmunoprecipi- tated with Cas from cultured podocytes transiently transfected with plasmid encoding CD16/7-nephrinCD (top) and from isolated rat glomerular cell lysate (bottom). (B) Human podocytes expressing CD16/7-nephrinCD (red) and Cas-GFP (green) were activated by clustering as in Figure 1, fixed, and imaged by confocal microscopy. (C) Cultured NIH3T3 cells expressing CD16/7-nephrinCD from the indicated plasmids were acti- vated as indicated. Immunoblots shown are representative of 3 independent experiments. (D) Cultured human podocytes were transfected with CD16/7-nephrinCD or CD16/7-HA (red) and activated, and endogenous p-Cas was identified using a specific p-Cas antibody and Alexa Fluor 488–conjugated secondary antibody. Cells were analyzed by confocal microscopy. (B and D) Original magnification, ×630. yz plane reconstruc- tions are shown at far right.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Phospho-proteomics, Cell Culture, Transfection, Plasmid Preparation, Isolation, Expressing, Confocal Microscopy, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 8 Crk is recruited to nephrin indirectly via Cas. Cas- null (A), Cas-WT (B), and Cas-15F (C) MEFs were transfected with CD16/7-nephrinCD and Crk-myc as indicated. Cells were activated by clustering as in Fig- ure 1 and analyzed by confocal microscopy. Crk-myc was stained with myc antibody and labeled with sec- ondary antibody (Alexa Fluor 488). Cells treated with PP2 prior to clustering are also shown. (A–C) Origi- nal magnification, ×630. yz plane reconstructions are shown at far right.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Transfection, Confocal Microscopy, Staining, Labeling

Journal: Journal of Clinical Investigation

Article Title: Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

doi: 10.1172/jci60070

Figure Lengend Snippet: Figure 9 Nephrin-induced actin filament polymerization and lamellipodia formation are mediated by distinct signaling pathways. (A) Nck1/2+/+ and Nck1/2–/– MEFs stably expressing CD16/7-nephrinCD were activated as in Figure 1, and endogenous p-Cas was stained with p-Cas antibody and Alexa Fluor 488–labeled secondary IgG antibody and imaged by confocal microscopy. Cells treated with PP2 prior to clustering are also shown. (B and C) Nephrin-induced lamellipodia formation was independent of Nck. Nck1/2+/+ and Nck1/2–/– MEFs expressing CD16/7-nephrinCD were evaluated for lamellipodial protrusion activity 30 minutes after clustering. Actin was stained with green-labeled phalloidin. (D) Cell lysates from Nck1/2+/+ and Nck1/2–/– MEFs were analyzed by immunoblotting with Nck antibody or GAPDH antibody as loading control. (E) Nephrin-induced local actin filament polymerization was independent of Crk1/2. Human podocyte cell lines stably expressing 1 of 5 different shRNA targeting human Crk1/2 were transfected with CD16/7-nephrinCD. Actin tails per podocyte were counted after activation by clustering. (A and B) Original magnification, ×630. (C and E) Data (mean ± SEM) are from 3 independent experiments. *P < 0.05; ***P < 0.0005.

Article Snippet: Monoclonal antibodies against Cas (BD Transduction), Crk (BD Transduction), CD16 clone 3G8 (BD Pharmingen), β-actin (Sigma-Aldrich), ZO-1 (Zymed), GST (BD Transduction), GAPDH (abcam), synaptopodin (GenWay), and phosphotyrosine clone 4G10 (Millipore) and polyclonal antibodies against p-Cas (Cell Signaling Technology), p-FAK (Cell Signaling Technology), c-myc (SigmaAldrich), and Nck (BD Transduction) were obtained commercially.

Techniques: Protein-Protein interactions, Stable Transfection, Expressing, Staining, Labeling, Confocal Microscopy, Activity Assay, Western Blot, Control, shRNA, Transfection, Activation Assay