Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 1. CCR2 is required for efficient SFTSV infection in cells. (A to C) RT-qPCR (A), flow cytometry (B), and Western blot (C) analysis of SFTSV infection at 24 hours after infection in CCR2- or ATF6-knockdown THP-1 cells. n = 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) RT-qPCR analysis of virus RNA in CCR2-KO THP-1 cells inoculated with four phylogenetically distinct SFTSV strains, including HBMC16, HNXY2017-50, HNXY2017-66, and WCH, for 24 hours. n = 4. (E) Multistep growth curves of four SFTSV strains in CCR2-KO THP-1 cells. n = 4. (F) Surface expression of CCR2 on BMDMs from CCR2−/−and WT C57BL/6J mice. A representative of three replicates is shown. (G and H) Statistical results (G) and scanned images (H) of the immunological focus assay of SFTSV titers at 24 hours after infection in BMDMs with deletions in CCR2. n = 6. (I) Microscopy of BMDMs immunostained for F4/80, SFTSV NP, and DAPI at 24 hours after infection. (J) SFTSV infection rates at 24 hours after infection determined by flow cytometry analysis in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (K) Representative flow plot of SFTSV infection in Huh7 cells. (L) Supernatant viral titers measured by immunological focus assay in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. One-way ANOVA followed by Tukey’s multiple comparisons test was per- formed for comparison of variables among three groups [(J) and (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Flow Cytometry, Western Blot, Knockdown, Virus, Expressing, Microscopy, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 2. Effect of CCR2 inhibitor and antibody on SFTSV infection in cells. (A to F) Effects of CCR2 antagonist RS102895 and CCR2 antagonist 1 on SFTSV infection in THP-1 (A) to (C) and Huh7 cells (D) to (F). At 24 hours after infection, relative vRNA levels were measured via RT-qPCR [(A), (B), (D), and (E), n = 3], and relative intracellular SFTSV NP levels were measured by Western blotting (C) and (F). Cell viability was measured using CCK-8 [(D) and (E), n = 3]. (G to I) Effects of CCR2 antibody on SFTSV infection in THP-1 cells. The dose-dependent inhibitory effects of the CCR2 antibody were analyzed by detecting virus loads in THP-1 cells at 24 hours after infection [(G), n = 6]. SFTSV infection rates were measured at 24 hours after infection with MOIs of 1 and 5 [(H), n = 6]. Representative flow plot of SFTSV infection in THP-1 cells treated with anti-human CCR2 or isotype control antibody (I). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), (E), (G), and (H)]. R2 [(A), (B), (D), and (E)] was estimated by a nonlinear regression model (curve fit).

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Virus, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 3. CCR2 mediates SFTSV binding and internalization. (A and B) Effects of CCR2 deletions on the internalization (A) and binding (B) of SFTSV in BMDMs. Relative vRNA levels were measured via RT-qPCR. n = 6. (C) Fluorescence microscopy analysis of the effects of CCR2 deletions on the binding of SFTSV. BMDMs were immunos- tained with F4/80 (green), SFTSV NP (red), and DAPI. A representative of three replicates is shown. (D and E) SFTSV binding assay in CCR2-KO (KO) THP-1 cells by using RT- qPCR (D) and immunofluorescence staining (E). n = 4. Cellular membranes were labeled with WGA. (F) Flow cytometry analysis of SFTSV infection at 2 hours after infection in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (G to I) Effects of the CCR2 antagonist RS102895 (G), CCR2 antagonist 1 (H), and favipiravir (I) on the binding and internalization of SFTSV in THP-1 cells. n = 3. (J) Binding assay of HRTV, RVFV, and AMRV for control and CCR2-KO THP-1 cells. n = 3. (K and L) Effects of CCR2 antagonist RS102895 (K) and CCR2 antagonist 1 (L) on the binding of HRTV, RVFV, and AMRV in THP-1 cells. n = 3. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (J)]. One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(F) to (I), (K), and (L)]. ns, no significance; p.i., post-infection.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling, Flow Cytometry, Infection, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 4. The CCR2 N-terminal extracellular domain mediates SFTSV binding to cells. (A) Coimmunoprecipitation of co-overex- pressed Gn-strep protein and CCR2-flag proteins in HEK293T cells using strep-tag binding beads or anti-Flag antibody beads. Vector-flag, GFP-flag, and SCARB1-flag pro- teins were used as negative controls. (B to D) Effect of CCR2 overexpression on the in- fectivity of SFTSV in Huh7 (B), HeLa (C), and Jurkat (D) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry analysis in control-, CCR2A-, CCR2B-, CCR2A-ΔN–, and CCR2B-ΔN–over- expressing cells. N = 6. (E) Surface expres- sion of CCR2 and representative flow plot of SFTSV infection in Jurkat cells. (F and G) Effect of the CCR2 N-terminal extracellular domain on the infectivity of SFTSV in HeLa (F) and Jurkat (G) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry in control-, CCR2A-, CCR2A-N14Q–, CCR2A-Y26F–, CCR2B-, CCR2B-N14Q–, and CCR2B-Y26F–overex- pressing cells. n = 6. (H) Binding of CCR2 N- terminal–derived Y26 sulfated peptide (p2), peptide without tyrosine sulfation at Y26 (p1), or the scrambled peptide to SFTSV virions determined by PRM assay. Statistical analysis is shown in the left panel, and PRM transitions are shown in the right panel. (I and J) Effect of CCR2 N-terminal–derived peptides on the infectivity of SFTSV in THP-1 cells. Relative intracellular vRNA levels and supernatant viral titers were measured at 24 hours after infection via RT-qPCR. n = 4. (K) Effect of CCR2 N-terminal–derived peptides on the binding of SFTSV in THP-1 cells. Rel- ative levels of bound virions were measured via RT-qPCR. n = 4. One-way ANOVA fol- lowed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(B) to (D) and (F) to (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Strep-tag, Plasmid Preparation, Over Expression, Infection, Flow Cytometry, Control, Expressing, Derivative Assay, Quantitative RT-PCR, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 5. CCR2 contributes to SFTSV pathogenesis in mouse models. (A) Serum and spleen viral titers in SFTSV-infected CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (B and C) Survival probability (B) and relative body weight (C) in anti-IFNAR1 antibody–pretreated CCR2−/−(n = 11) and WT (n = 11) C57BL/6J mice after intraperitoneal infection with SFTSV. (D) Viral titers in serum, spleen, liver, and lung samples from anti-IFNAR1 antibody–pre- treated CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (E and F) Representative images of spleen, liver, and lung sections collected at 5 dpi from control and SFTSV-challenged CCR2−/−and WT C57BL/6J mice stained with a rabbit polyclonal antibody against SFTSV NP (E) or with hematoxylin and eosin (F). (G) Viral titers in serum from anti-IFNAR1 antibody–pretreated C57BL/6J mice tested by immunological focus assay at 3 (n = 13 for each group) and 5 (n = 10 for nontreated group and n = 13 for treated group) dpi. (H) Survival probability in anti-IFNAR1 antibody–pretreated C57BL/6J mice with SFTSV infection in the absence (n = 13) or presence (n = 13) of CCR2 antagonist RS102895 and without SFTSV infection (n = 5). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. The Kaplan-Meier method was used to analyze time-to-event data [(C) and (H)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Control, Staining, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 6. CCR2 contributes to SFTSV infectivity in primary human monocytes. (A and B) Comparisons of surface CCR2 expression levels on primary human mono- cytes (left) and the ability of SFTS binding (right) between donors aged <60 (n = 10) and ≥60 (n = 10) years old (A), as well as between healthy donors (n = 8) and donors with DM (n = 8) (B). MFI, mean fluorescent intensity. (C) Association of surface CCR2 expression level on primary human monocytes with virus binding ability or with the age of donors (n = 20). Ra 2 indicates the correlation between the CCR2 expression level and individual age, and Rb 2 indicates the correlation between the CCR2 expression level and the binding ability of SFTSV. (D) Associa- tion of surface CCR2 expression level on primary human monocytes with the peak viral load in serum that was consecutively collected from SFTS patients (n = 45) during the clinical course. Two-tailed Student’s t test was performed for compar- ison of variables between two groups [(A) and (B)]. R2 [(C) and (D)] was estimated by a linear regression model. HC, healthy control; DM, diabetes mellitus; CT, cycle threshold.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Expressing, Binding Assay, Virus, Two Tailed Test, Control

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Mural cells sustain a vascular MΦ niche (A) In vivo multi-photon imaging of Ca 2+ signal and morphological changes of MΦs in Cx3cr1-MΦ Ca-rep mice in an environment of laser-induced microinjuries. MΦs are depicted in red; Ca 2+ signal and vascular flow are depicted in green. MΦs have been rendered additionally below, with a pseudocolored depiction of the Ca 2+ signal. Images are derived from . (B) In vivo and ex vivo confocal and airy-scan imaging of MC-MΦ contacts across organs in MC RFP-rep ; Cx3cr1-MΦ GFP-rep mice, arrows depicting cell-cell contacts: top left: intravital imaging of the microvasculature in the mesentery, the dashed line is depicting MCs (scale bars, 5 μm); top middle: ex vivo imaging of the heart microvasculature (scale bars, 50 μm); top right: en face ex vivo imaging of the aortic atherosclerotic intima after 3 months of western-diet feeding (macrovasculature), the dashed line is subdividing the plaque core from the shoulder region (scale bars, 10 μm); bottom left: ex vivo imaging of the kidney microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 7 μm); bottom middle: ex vivo imaging of the lung microvasculature (scale bars, 20 μm), including higher magnification below (Cx3cr1 hi CD68 lo interstitial MΦs (iMΦs) in green, CD68 hi Cx3cr1 lo alveolar MΦs (aMΦs) in white, MCs in red) (dashed line depicting MCs) (scale bars, 5 μm); bottom right: ex vivo imaging of the stomach microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 10 μm). Interstitial MΦs are shown in green, and MCs are shown in red for all organs, with further subdifferentiation of MΦs in the lung (as depicted above). (C) Analysis of the time until MΦs form their first dendrites (left) and time which MΦs require to reach injury (right), as the time in minutes after laser injury, in MΦ GFP-rep mice treated locally (subcutaneously) and systemically with isotype or CCL2-neutralizing antibody (n = 17–37 individual cells analyzed from 3 to –4 mice/group). (D) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . Uniform Manifold Approximation and Projection (UMAP) based dimensionality reduction of analyzed cells. (E) Highly expressed cytokines and chemokines in human SMCs from coronary arteries analyzed from cells shown in (D) CCL2 is highlighted as the most prominently expressed chemokine. (F) Percentage of peritoneal macrophage survival upon CCL2 stimulation at different time points under starvation stress conditions (n = 3 experiments). (G) Quantification of CD68 + perivascular macrophage content in Ccl2 MC+/+ and Ccl2 MCΔ/Δ mice in percentage of total perivascular area (15 μm radius around the vessel) in the kidney (n = 5–6 mice/group). (H) Quantification of cell proliferation as EdU + cells relative to CD68 + area (as number of proliferating cells/μm 2 ). (I) Quantification of blood monocyte counts by automated blood counter (n = 5–6). (J) Representative images from immunofluorescence staining of kidney sections in Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice for ACTA2 (red), CD68 (green). Scale bars, 50 μm (left: Ccl2 MC+/+ ; right: Ccl2 MCΔ/Δ ). (C, G, H, and I) Student’s t test was used. (F) Repeated measures two-way ANOVA was -808990139890500used. ∗ p < 0.05. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: In Vivo, Imaging, Derivative Assay, Ex Vivo, Western Blot, Single Cell, RNA Sequencing, Immunofluorescence, Staining

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: MC-derived CCL2 sustains a homeostatic MΦ phenotype across the vascular tree (A and B) UMAP based dimensionality reduction of single-cell RNA-seq of FACS-sort enriched CD45 + CD11b hi CD64 hi F4/80 hi cells in kidney (A) and lung (B) of Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice (n = 4/group). (C–G) Volcano and violin plots depicting selected significantly differentially regulated genes in (C) kidney resident MΦ cluster 2, (D) kidney resident MΦ cluster 0, (E) lung monocyte cluster 1, (F) lung alveolar MΦ cluster 2, (G) lung Folr2 hi Mrc1 hi interstitial MΦ cluster 3. (H) Frequency of lung Zeb2 hi interstitial MΦ cluster 4 cells among all analyzed cells. (I) Significantly differentially regulated genes, associated with a functionally differentiated, efferocytotic MΦ phenotype in Ccl2 MC+/+ and Ccl2 MCΔ/Δ chimera mice. Low-input RNA-seq of FACS-sorted Cx3cr1 + MΦs from Ccl2 MC+/+ or Ccl2 MCΔ/Δ chimera mice with MC RFP-ep ; Cx3cr1-MΦ GFP-rep bone marrow after 20 weeks western diet (experimental setup further depicted in Figure S3 ) (n = 3–4 chimera mice). Student’s t test was used. Expression levels of depicted genes normalized to sample with highest expression (set as 1) across all samples. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression. ∗ p < 0.05.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Derivative Assay, Single Cell, RNA Sequencing, Western Blot, Expressing

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Distinct chemotactic SMCs express high levels of MΦ chemoattractants, ameliorating atheroprogression (A–C) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . (A) Violin plots (calculated on all cells expressing detectable baseline levels of the respective gene) of highly expressed cytokines and chemokines in chemotactic SMCs. Dots represent single cells, only cells exhibiting detectable expression of the particular gene are included (B) interactome depicting cell-cell interactions between MΦ and SMC subsets, prominent SMC → MΦ interactions are depicted in red. Intensity of red color depicts the respective portion of the CCL2-CCR2 axis for the concrete interaction (the darker the red color, the more the CCL2-CCR2 axis accounts for the respective inter-cluster interplay among all detected chemokine-receptor interactions). (C) Heatmap further unraveling SMC → MΦ chemokine:chemokine-receptor interactions. Blue box depicts interactions of chemotactic SMC subset, red box depicts CCL2-mediated interactions between SMC and MΦ subsets. (D) Ccl2 and Mif expression in Ng2 + SMCs FACS-sorted from western-diet fed atherosclerotic MC RFP-rep mice compared to chow-diet fed non-atherosclerotic control mice. n = 3–4 mice per group. (E) Representative images of BCA sections from Ccl2 SMC +/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet stained for ACTA2 (green), LGALS3 (red), and Hoechst (blue). Scale bars, 100 μm. (F and G) Morphometric analysis of plaque size (F) and vascular remodeling (G) from BCA sections at three consecutive locations from Ccl2 SMC +/+ (n = 11) and Ccl2 SMCΔ/Δ (n = 10) littermates. (H and I) Quantification of ACTA2 + smooth muscle cell content as ACTA2 + area in percentage of total plaque area and percentage of 30 μm plaque surface area in valves (H) and in the BCA at three consecutive locations (I). (H and I) n = 10–11 mice per group. (J and K) Analysis of intimal LGALS3 + area as percentage of plaque size in BCA sections at three consecutive locations (J) and in plaques from aortic valves (K) (n = 10–11 each). (L) Schematic illustration of media and intima processing from aortae of Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet (left). Heatmap displaying expression of differentially regulated genes in bulk RNA-seq of Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4). Rows represent individual replicates, differentially expressed genes are illustrated in columns (right). (M) Volcano plots of intima/media RNA-seq showing differentially expressed genes in Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4), x-axis depicts Log2FC, y-axis depicts -Log10(adj. p-value). Data are shown as mean and SEM. (H and K), Student’s t test was used. (F, G, I, and J) Repeated measures two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; NS, not significant. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Control, Staining

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: SMCs within the fibrous cap preserve a strategic positioning of plaque MΦs and secure homeostatic MΦ functions (A and B) Reanalyzed single-cell RNA-seq data from mouse aortic roots from atherosclerotic SMC lin mice from Wirka et al., GEO: GSE131780 . (A) UMAP based dimensionality reduction of analyzed cells (left), heatmap illustrating cytokine and chemokine expression of different SMC subsets (right). (B) Marker genes of SMC clusters illustrated in a heatmap, composed by ClustVis. (C) Representative confocal image depicting the spatial distribution of the key cSMC marker PDGFRβ within an atherosclerotic valve in SMC lin ; Cx3cr1-MΦ GFP-rep mice after 22–24 weeks of western diet, SMC lin cells in red, MΦs in green, and PDGFRβ in white. Scale bars: 30 μm (left) and 15 μm (right images). (D) Illustration of the experimental setup of the migration assay: macrophages undergo a migratory decision either moving toward the artificially composed SMC-rich fibrous cap below or residing at the artificially composed, necrotic cell rich, necrotic core. SMCs (representing the fibrous cap) are located in the lower chamber, whereas peritoneal macrophages have been attached on the transwell of the upper chamber. Necrotic Jurkat cells (representing the necrotic core) have been added to the upper chamber. (E) Number of peritoneal MΦs from Lyz-MΦ GFP-rep mice that transmigrated toward the lower chamber per field of view (FOV). Isotype or anti-CCL2 blocking antibody was simultaneously added to the lower chamber. MΦ numbers per FOV counted at 4 subsequent time points (n = 4 independent experiments). (F) Distribution of macrophages as percentage of LGALS3 + area in 30 μm plaque surface area in percentage of total plaque LGALS3 + area at three subsequent locations (n = 10 each). (G) Left: quantification of LGALS3 + surface macrophage content as relative LGALS3 + area in percentage of total plaque surface area (defined as the upper 30 μm of the plaque) from BCA sections at three consecutive locations (n = 10 each). Right: representative immunofluorescent images of BCA sections for ACTA2 (green), LGALS3 (red), and Hoechst (blue) with highlighted 30 μm plaque surface area from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western-diet feeding. Scale bars, 100 μm. (H) Volcano plot depicting differentially regulated genes analyzed by RNA-seq of FACS-sort enriched peritoneal MΦs, coincubated either with live or dead Jurkat cell supernatant for 12 h. (I) Quantification of peritoneal macrophages 12 h after addition of live or dead Jurkat cell supernatant (n = 6). (J–L) Efferocytosis assay, analyzing the efferocytotic capacity of the MΦ population, isolated from Lyz-MΦ GFP-rep mice. Apoptotic Jurkat cells were added for 1 h after 6 h incubation either with or without CCL2. (J) Quantification of MΦs with engulfed apoptotic cells upon presence or absence of CCL2 (n = 5 independent experiments). (K) Quantification of the total number of engulfed apoptotic cells upon CCL2 presence of absence. (L) Representative epifluorescence images of the efferocytosis assay with peritoneal macrophages (green) and apoptotic Jurkat cells (red), 1 h after Jurkat cell addition. Scale bars, 50 μm. (M–O) Necrotic core analysis as total necrotic area in μm 2 (M) and in percentage of plaque area (N), assessed with Masson Trichrom’s staining of valve sections, from Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) littermates after 14 weeks of western diet. (O) Left: representative images of necrotic core content analyzed by Masson Trichrom’s staining of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet. ∗ indicates necrotic areas. Scale bars, 100 μm. Right: representative images of immunofluorescence stainings of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet for ACTA2 (green), LGALS3 (red), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (yellow), and DAPI (blue). Scale bars, 100 μm. (P and Q) Quantification of cell apoptosis as total amount of TUNEL + LGALS3 + Hoechst + MΦs in plaque (P) and as total amount of TUNEL + Hoechst + apoptotic cells (Q) in Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) individual littermates in total after 14 weeks of western diet, only including plaques at the proximal and intermediate BCA, without distal BCA areas with its early lesions. (R) Quantification of valve atherosclerotic plaques for (left) total and relative TUNEL + cells. Data are shown as mean and SEM. (I, J, K, M, N, and R) Student’s t test was used for normally distributed data and Wilcoxon matched-pairs signed rank test for not normally distributed data. (E, F, G, P, and Q) Repeated measure two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; ∗∗ p < 0.01; NS, not significant. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Western Blot, Migration, Blocking Assay, Isolation, Incubation, Staining, Immunofluorescence, End Labeling, TUNEL Assay

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Short-term CCL2 inhibition in advanced atherosclerosis triggers detrimental changes in plaque phenotype (A) Acute pharmacological CCL2 inhibition in ApoE −/− mice after 6 months of western type diet. The anti-CCL2 or isotype control antibody was injected intravenous (i.v.) 2 weeks before sacrifice every 48 h (n = 7–8 / group). (B) Quantification of fibrous cap coverage as continuity (percentage of fibrous cap covered plaque surface length relative to complete plaque surface length) at three subsequent BCA locations. (C) Quantification of ACTA2 + area within plaque surface as % of plaque surface area (defined as the top 30 μm stripe of the lesion) at three subsequent BCA locations. (D) Quantification of absolute ACTA2 + area in μm 2 at three subsequent BCA locations. (E) Quantification of macrophage area as LGALS3 area in μm 2 at three subsequent BCA locations. (F) Quantification of total plaque size as absolute plaque area in μm 2 at three subsequent BCA locations. (G) Quantification of cell apoptosis as total amount of TUNEL + cells in plaque at three subsequent BCA locations. (H) Representative images of BCA sections from ApoE −/− mice after 6 months of western diet stained for ACTA2 (green), LGALS3 (far red), TUNEL (red), and Hoechst (blue). Scale bars, 50 μm. (I) Quantification of blood leukocytes, neutrophils, lymphocytes, monocytes, and plasma cholesterol (n = 7–8). (I) Student’s t test was used. (B–G) Repeated measures two-way ANOVA or mixed-effects model, with subsequent Šídák’s multiple comparisons test in (B)–(D), was used. ∗ p < 0.05; ∗∗ p < 0.01 NS, not significant. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Inhibition, Western Blot, Control, Injection, TUNEL Assay, Staining, Clinical Proteomics

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet:

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Control, Blocking Assay, Selection, Recombinant, Western Blot, In Situ, Staining, Single Cell, DNA HS Assay, Picogreen Assay, cDNA Synthesis, Clinical Proteomics, SYBR Green Assay, Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Software

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 mediates the regulation of chemokine biosynthesis in M1-polarized macrophages, as determined by RNA-seq. ( A ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h and subjected to RNA sequencing analysis. The KEGG database was used to identify enriched signaling pathways whose activation significantly differed (up). Single-gene GSEA was conducted using the GSE57065 , GSE65682 , and GSE95233 datasets to identify the relationship between PD-L1 and chemokine signaling pathways (down). ( B ) Intersection analysis of upregulated genes between the PD-L1 HI /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + LPS + IFN-γ group and between the PD-L1 NC /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + PBS group via a Venn diagram, followed by further intersection with chemokine datasets, revealed three highly expressed PD-L1-mediated chemokines (CCL8, CXCL9, and CXCL10) under LPS + IFN-γ stimulation. ( C ) RNA sequencing analysis revealed the expression of CCL8, CXCL9 and CXCL10 upon PD-L1 overexpression. ( D ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( E ) Human monocytes were induced into macrophages, transfected with scramble siRNA and PD-L1 siRNA, and then treated with LPS + IFN-γ for 12 h. CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( F ) PD-L1 NC /THP-1-M and PD-L1 KO /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for D, E and F was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: RNA Sequencing, Protein-Protein interactions, Activation Assay, Expressing, Over Expression, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: TLR4 acts as a critical upstream regulator of PD-L1 in modulating CCL8 and CXCL9 expression. ( A ) GSEA of RNA-seq data to investigate the relationship between PD-L1 expression and Toll-like receptor signaling pathway activation. ( B ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TLR4 siRNA, respectively, followed by 12 h of stimulation with LPS (100 ng/mL) + IFN-γ (20 ng/mL). The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, RNA Sequencing, Activation Assay, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 binds to the TLR4 downstream molecule TRAF6 to regulate CCL8 and CXCL9 expression. ( A ) Venn diagram analysis of proteins coimmunoprecipitated with PD-L1 in THP-1-M cells stimulated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h via IP‒MS. After excluding proteins bound to the IgG control group, 289 PD-L1-specific interacting proteins were identified (up). Representative MS/MS spectrum showing peptide fragments of the TRAF6 protein identified in the immunoprecipitation complex (down). ( B ) THP-1-M (up) and human monocyte-derived (down) macrophages were treated with or without LPS+IFN-γ for 6 h and then harvested for Co-IP. TRAF6 protein levels were measured via Western blotting. ( C ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TRAF6 siRNA, respectively, followed by 12 hours of stimulation with LPS+IFN-γ. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). ( D ) Structural models of full-length PD-L1 and TRAF6 predicted by AlphaFold (PAE plots were obtained from AlphaFold). ( E ) Molecular docking generic matching information. ( F ) A protein‒protein interaction cartoon model generated by PyMOL (left). Predicted binding sites between PD-L1 and TRAF6 (right). ( G ) PD-L1 NC /THP-1-M, PD-L1 HI /THP-1-M and PD-L1 Δ62-139 /THP-1-M cells were treated with LPS+IFN-γ for 12 h. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C and G was performed by one-way ANOVA with LSD test for post hoc analyses. * p <0.05.

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, Control, Tandem Mass Spectroscopy, Immunoprecipitation, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Generated, Binding Assay

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 knockout attenuates septic mouse mortality and suppresses CCL8 and CXCL9 expression. ( A – C ) PD-L1 WT /mice and PD-L1 KO /mice were intraperitoneally injected with LPS (15 mg/kg) and IFN-γ (10 µg/kg) to induce endotoxemia-induced sepsis. ( B ) The survival rate was determined over a 60-hour observation period and is presented as a Kaplan‒Meier survival curve ( n = 15 mice per group). The results are presented as Kaplan–Meier survival curves. The results of the statistical analysis were analyzed with the log-rank test. * p < 0.05. ( C ) Peripheral blood was collected 12 h posttreatment for serum isolation and PBMC separation. CCL8 and CXCL9 protein levels in serum and their corresponding mRNA levels in PBMCs were subsequently analyzed ( n = 3 independent biological replicates). ( D ) Peritoneal macrophages (PMs) were extracted from PD-L1 wild-type and knockout mice and then treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). (E) Peripheral blood monocytes were extracted from PD-L1 wild-type and knockout mice and then treated with LPS + IFN-γ for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C was performed by Student’s t test, and that for D and E was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Knock-Out, Expressing, Injection, Isolation

Journal: Journal of Translational Medicine

Article Title: Single-cell RNA sequencing reveals recruitment of the M2-like CCL8 high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

doi: 10.1186/s12967-024-05118-6

Figure Lengend Snippet: Single-cell transcriptomic landscape of subcutaneous LLC tumors at early-stage following hypofractionated radiotherapy. A Overview of the experimental design for single-cell RNA sequencing. B Uniform Manifold Approximation (UMAP) plot showing the unsupervised clusters of 54883 single cells and annotated cell types. C The proportion of each cell types in LLC tumors treated with or without radiation. For B and C, each color represents the same cell type. D Feature plot and E Dot plot showing marker genes. F Heatmap showing serum chemokines level in LLC murine models 72 h post-treatment. Each row in the heatmap has been scaled. G The concentrations of CCL2, CCL7, CCL8, and CSF1 in serum measured by ELISA (n = 5/group). Data are presented as mean ± SD with student unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant

Article Snippet: For proliferation assay and colony formation assay, the LLC cell line was treated with recombinant CCL8 (MedChemExpress, Cat#: HY-P7771) at 0, 5 ng/ml, 20 ng/ml.

Techniques: RNA Sequencing, Marker, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Single-cell RNA sequencing reveals recruitment of the M2-like CCL8 high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

doi: 10.1186/s12967-024-05118-6

Figure Lengend Snippet: Identification of M2-like Ccl8 high Macrophages in the LLC-bearing murine model. A UMAP plot showing the annotation of macrophage populations in LLC tumors. B Feature plot and C Dotplot showing marker genes of macrophage populations. D Cell proportion of each cell type in NT and RT groups. E Heatmap displaying scores of M1, M2, angiogenesis, phagocytosis for each macrophage population. F Correlation analysis of CCL8 and CD163 expression level in TCGA-LUAD datasets (Spearman’s rho value = 0.542, p < 0.001). G The Gene Ontology (GO) enrichment analysis of each macrophage populations. H Heatmap displaying transcription factors activity of the Mac_Ccl8, Mac_Hmox1, Mono_Cxcl3, and Mono_Plac8 populations. I Development trajectory of macrophages populations predicted by Monocle2. J The cell density and K gene expression patterns along with the pseudotime,

Article Snippet: For proliferation assay and colony formation assay, the LLC cell line was treated with recombinant CCL8 (MedChemExpress, Cat#: HY-P7771) at 0, 5 ng/ml, 20 ng/ml.

Techniques: Marker, Expressing, Activity Assay, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Single-cell RNA sequencing reveals recruitment of the M2-like CCL8 high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

doi: 10.1186/s12967-024-05118-6

Figure Lengend Snippet: Hypofractionated radiotherapy promoted the crosstalk between the Mac_Ccl8 and lymphocytes. A UMAP plot showing the annotation of lymphocyte populations. B Feature plot and C Dot plot showing marker genes of lymphocyte populations. D The proportion of each lymphocyte population in two groups. E Violin plots displaying the expression level of immune checkpoint ligand genes in each lymphocyte populations of NT and RT groups. F Circle plots showing number of interactions in two groups inferred by CellChat. G The comparison of cellular communication probability from Mac_Ccl8 to T and NK cells in between two groups. H Chord plot displaying the upregulated signaling pathways in the RT group relative to the NT group. I Heatmap of the differential interaction strength of the GALECTIN signaling pathway between two groups

Article Snippet: For proliferation assay and colony formation assay, the LLC cell line was treated with recombinant CCL8 (MedChemExpress, Cat#: HY-P7771) at 0, 5 ng/ml, 20 ng/ml.

Techniques: Marker, Expressing, Comparison, Protein-Protein interactions

Journal: Journal of Translational Medicine

Article Title: Single-cell RNA sequencing reveals recruitment of the M2-like CCL8 high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

doi: 10.1186/s12967-024-05118-6

Figure Lengend Snippet: Hypofractionated radiotherapy reprograms CCL8 high macrophages through the CCL signaling pathway. A Volcano plot showing differentially expressed genes of the Mac_Ccl8 between the NT and RT groups. Adjusted p value < 0.05, two-sided Wilcoxon test. B Violin plots comparing the expression of Ccl2, Ccl3, Ccl4, Ccl7, Ccl8, and Ccl12 in the NT and RT groups. Unpaired two-sided Wilcoxon test. C Representative examples of multiplex immunofluorescent labeling CD206 and CCL8. Green, CD206; Red, CCL8; Blue, DAPI. D Bar plots showing the GO enrichment analysis of upregulation and downregulation genes in the RT group. E Differences in IFN-Gamma and TNF pathways activity between two groups inferred by GSEA. F Cell–cell communication network between myeloid populations and LLC cells. G River plot displaying communication patterns of different cell types. H Chord plot (top) and heatmap (bottom) showing communication network of CCL signaling pathway in different cell types. I Weighted network analysis of differential interaction strength of signals in the Mac_Ccl8 population between two groups. J The comparison of cellular communication probability from Mac_Ccl8 to other myeloid populations between two groups. K Chord plot displaying the upregulated signaling pathways in the RT group relative to the NT group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant

Article Snippet: For proliferation assay and colony formation assay, the LLC cell line was treated with recombinant CCL8 (MedChemExpress, Cat#: HY-P7771) at 0, 5 ng/ml, 20 ng/ml.

Techniques: Expressing, Multiplex Assay, Labeling, Activity Assay, Comparison, Protein-Protein interactions

Journal: Journal of Translational Medicine

Article Title: Single-cell RNA sequencing reveals recruitment of the M2-like CCL8 high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

doi: 10.1186/s12967-024-05118-6

Figure Lengend Snippet: Hypofractionated radiotherapy promotes M2-like Ccl8 high macrophages infiltration and leads to poor prognosis. A Kaplan–Meier plots showing worse clinical prognosis in the LUAD and HNSC patients with the higher expression level of the Mac_Ccl8 signature. HR, hazard ratio. B Schematic diagram of the combination treatment with hypofractionated radiotherapy and the Bindarit. The intraperitoneally administration of Bindarit began from day 5 to day 11 post-tumor injection, and radiation treatment was initiated from day 7 to day 9 post-tumor injection. C Growth curves of tumors in LLC-bearing mice in the indicated treatment groups (n = 5 mice/group). Data are presented as mean ± SD with two-way ANOVA test. D Representative examples in the indicated treatment groups of multiplex immunofluorescent labeling F4/80, CD206 and CCL8. Yellow, F4/80, Green, CD206; Red, CCL8; Blue, DAPI. E Percentages of M1 and M2 macrophages in the specific treatment groups analyzed by flow cytometry (n = 3/group). F Representative flow cytometry panels showing M2 macrophages (top) and M1 macrophages (bottom). BI, Bindarit; RT, Radiation therapy; RT + BI, the combination therapy of the radiation and the Bindarit

Article Snippet: For proliferation assay and colony formation assay, the LLC cell line was treated with recombinant CCL8 (MedChemExpress, Cat#: HY-P7771) at 0, 5 ng/ml, 20 ng/ml.

Techniques: Expressing, Injection, Multiplex Assay, Labeling, Flow Cytometry

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Construction and bioactivity evaluation of WEV-bFGF. ( A ) Spatial structure prediction of WEV-bFGF recombinant protein and natural bFGF; ( B ) SDS-PAGE of bFGF, KIT-bFGF and WEV-bFGF; ( C ) Western blot of native bFGF, KIT-bFGF and WEV-bFGF; ( D ) The biological activities of native bFGF, KIT-bFGF and WEV-bFGF by CCK-8 assay; ( E ) The fluorescence distribution of Delight 800 labeled recombinant proteins in hypoxic HK-2 cells; ( F ) Statistical analysis of fluorescence intensity of Dylight 800 in hypoxic HK-2 cells; Scale bar = 25 μm; ( G ) The protective effects of recombinant proteins on HK-2 cells after hypoxic injury by the CCK8 assay. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Recombinant, SDS Page, Western Blot, CCK-8 Assay, Fluorescence, Labeling

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: The evaluation of targeting capacity of WEV-bFGF in ischemic kidney in vivo . ( A ) At 6 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( B ) Western blot showing bFGF protein expression in ischemic renal tissue 6 h after administration; ( C ) Quantitative analysis of relative bFGF protein expression at 6 h; ( D ) The content of bFGF in ischemic kidneys by ELISA assay at 6 h after administration; ( E ) The content of bFGF in serum at 6 h after administration; ( F ) Immunofluorescence colocalization of bFGF and KIM-1 in the frozen sections at 6 h after administration, Scale bar = 50 μm; ( G ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 6 h after administration; ( H ) At 24 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( I ) Western blot showing bFGF protein expression in ischemic renal tissue 24 h after administration; ( J ) Quantitative analysis of relative bFGF protein expression at 24 h; ( K ) The content of bFGF in ischemic kidneys by ELISA assay at 24 h after administration; ( L ) The content of bFGF in serum at 24 h after administration; ( M ) Immunofluorescence staining showing colocalization of bFGF and KIM-1 at 24 h after administration, scale bar = 50 μm; ( N ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 24 h after administration. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: In Vivo, Fluorescence, Labeling, Recombinant, Imaging, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Functional tests and morphological analysis of the kidneys. ( A ) Experimental design of WEV-bFGF for the treatment of I/R; ( B ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( C ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( D ) H&E staining for kidney histopathological evaluation after I/R injury. Arrows indicated the sites of renal tubular injury. Scale bar = 20 μm; ( E ) Statistical analysis of renal tubular injury score; ( F ) MASSON staining for renal tissue fibrosis evaluation after I/R injury. Scale bar = 50 μm; ( G ) Statistical analysis of renal tissue fibrosis. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Functional Assay, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Transcriptome analysis of potential mechanism in WEV-bFGF mediating ischemic renal repair. ( A ) Volcano plots of gene differences between the WEV group and the PBS group; ( B ) KEGG analysis between the WEV group and the PBS group; ( C ) Volcano plots of gene differences between the WEV-BFGF group and the bFGF group; ( D ) KEGG analysis between the WEV-bFGF group and the bFGF group; ( E ) Heat map of differentially expressed genes related to repair and regeneration of AKI; ( F ) Quantitative PCR validation of key gene expression. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 6.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Gene Expression

Journal: EMBO Molecular Medicine

Article Title: Adaptively evolved human oral actinomyces‐sourced defensins show therapeutic potential

doi: 10.15252/emmm.202114499

Figure Lengend Snippet: An inhibition‐zone‐based assay for evaluating the stability of AMSIN in H 2 O or normal mouse serum. The bacterium used was BM and the peptide dose was 0.2 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (* P < 0.05, ns: no significance; exact P values are listed in Table ). Hemolysis by AMSIN. Meucin‐18 was used as a positive control. Mean ± SD of three technical replicates is displayed. Comparison of cytotoxic effects of AMSIN and LL‐37 on HL‐60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk; * P < 0.05, *** P < 0.001, ns: no significance; exact P values are listed in Table ). An inhibition‐zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (ns: no significance; exact P values are listed in Table ). The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady‐state current traces after administering 100 μM AMSIN. Peptide‐induced IL‐8 release. A549 cells were treated with AMSIN or LL‐37 for 24 h. IL‐8 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significance; exact P values are listed in Table ). Peptide‐induced MCP‐1 release. HL‐60 cells were treated with AMSIN or LL‐37 for 24 h. MCP‐1 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, ns: no significance; exact P values are listed in Table ). Surface plasmon resonance (SPR)‐based assay detecting anti‐AMSIN antibodies in mouse serum using Biacore T100. Sera were taken from three AMSIN‐treated mice (Samples 1–3) and one control mouse only injected with 0.9% NaCl. The sensorgrams present data of subtracting the background SPR signals from the control. Note: the abnormal peaks at 30 and 90 s yielded by the equipment due to sample injections were artificially removed. Insect, schematic diagram of SPR experiment in which 1:100 diluted serum was used as analyte to flow over the AMSIN‐immobilized CM5 chip. Treatment of pneumococcal pneumonia. Five mice per sampling point were inoculated with SP D39 through the nasopharynx and 24 h later the animals received AMSIN or penicillin (as a positive control) at different doses (AMSIN, at the doses of 8.35, 16.7, and 33 mg per kg; penicillin at 8.35, 16.7, and 30 mg per kg) via intramuscular injection (i.m.). One day after treatment, the animals were necropsied and their lungs were removed and homogenized for the determination of the level of SP , as described in the Materials and Methods section. Each point represents the determination from a single animal and the line shows the mean log value. Mean ± SD of five to ten biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by red asterisks; ** P < 0.01, *** P < 0.001 denotes significant reduction in CFU compared with the saline group; exact P values are listed in Table ). Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle‐treated control group was zero out of 11 mice and the treatment group contained six mice and all survived. Source data are available online for this figure.

Article Snippet: Medium was aspirated and replaced with fresh complete DMEM supplemented with 10% FBS or serum‐free medium, both containing different concentrations of polypeptides for 24 h. Then, the supernatants were harvested for quantification of the IL‐8 amount in A549 by the Human CXCL8/IL‐8 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) and the MCP‐1 amount in HL‐60 by the Human MCP‐1 ELISA Kit (Boster Biological Technology Co., Ltd, Wuhan, China) as per the manufactures’ instructions.

Techniques: Inhibition, MANN-WHITNEY, Positive Control, Comparison, Enzyme-linked Immunosorbent Assay, SPR Assay, Control, Injection, Sampling, Saline, Infection

Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated