human tarm1 cdna  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: SEMA3F dimerizes surface NRP2 and PLXNA1, inhibits U-251 MG cell proliferation, and reduces p-AKT level and CCND1 gene expression . A , effects of SEMA3F on proliferation of various cancer cell lines. Cells were treated with full-length SEMA3F for 3 days, and cell viability was measured using CellTiter-Glo, normalized to untreated cells. B , schematic illustrates the full-length SEMA3F dimer, the furin processing site, and the resulting furin-processed SEMA3F (SEMA3F-p65). C , SEMA3F-p65 does not inhibit U-251 MG cell proliferation compared to nonprocessed SEMA3F dimer. D , the anti-NRP2 antibody (aNRP2-a2) blocks SEMA3F-mediated inhibition of U-251 MG cell proliferation. E , schematic illustrating the receptor dimerization assay. NanoLuc luciferase is split into Large BiT and Small BiT with low activity, and fused to the N termini of NRP2 and PLXNA1. SEMA3F induces NRP2-PLXNA1 dimerization, bringing Large BiT and Small BiT together to enhance luciferase activity through complementation. F , SEMA3F treatment induces NRP2-PLXNA1 dimerization. Left panel : Expi293F cells coexpressing Large_BiT-NRP2 and Small_BiT-PLXNA1 were treated with luciferase substrate and SEMA3F as indicated, and luminescence was recorded over time. Right panel : dimerization ratio changes following SEMA3F treatment. G , SEMA3F reduces p-AKT levels in U-251 MG cells in an NRP2-dependent manner. U-251 MG cells were treated as indicated for 30 min, and the p-AKT/AKT ratio was measured. H , SEM3F downregulates CCND1 expression in an NRP2-dependent manner. U-251 MG cells treated for 18 h. The expression of CCND1 was quantified using qPCR and normalized to untreated control cells. I , schematic representation of SEMA3F’s anti-proliferative mechanism in U-251 MG cells. NRP, neuropilin; aNRP2, anti-NRP2; PLXNA1, plexinA1; SEMA, semaphorin; p-AKT, phosphorylation of AKT; qPCR, quantitative PCR.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Gene Expression, Inhibition, Luciferase, Activity Assay, Expressing, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Experimental workflow for the discovery of SEMA3F-mimetic bsAbs . MAb-targeting PLXNA1 (aPLXNA1) were generated and paired with anti-NRP2 antibodies to create bispecific antibodies (bsAbs). These bsAbs were initially evaluated in a PLXNA1-NRP2 dimerization assay to identify candidates that effectively promote PLXNA1–NRP2 interaction. Positive bsAbs were then assessed using a p-AKT assay, a qPCR assay for CCND1 expression, and a cell viability assay to determine which candidates mimic SEMA3F’s effects. Ultimately, one bsAb, P1943-Nb2cL, was identified as mimicking SEMA3F’s effects across all cell-based assays. aPLXNA1, anti-PLXNA1; NRP, neuropilin; PLXNA1, plexinA1; SEMA, semaphorin; p-AKT, phosphorylation of AKT; qPCR, quantitative PCR.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Generated, Expressing, Viability Assay, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Discovery of anti-PLXNA1 mAbs . A , SDS-PAGE analysis of purified PLXNA1-ECD and PLXNA1-LBD proteins used for mouse immunization. The gel displays nonreduced (without reducing agent in the loading buffer) and reduced (with DTT added to the loading buffer) conditions. B , schematic illustrating the mouse immunization schedule, detailing the timeline for immunization, serum collection, and cell harvest. C , ELISA-based quantification of serum antibody levels following booster immunizations. Data represents six mice immunized with either PLXNA1-ECD or PLXNA1-LBD proteins. D , analysis of germline heavy chain IGHV gene usage in anti-PLXNA1 antibodies. A total of 135 cloned antibodies were analyzed, revealing usage of 29 distinct heavy chain IGHV genes. The pie chart shows the distribution of antibodies based on their IGHV gene usage. ECD, extracellular domain; LBD, ligand-binding domain; PLXNA1, plexinA1; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: SDS Page, Purification, Enzyme-linked Immunosorbent Assay, Clone Assay, Ligand Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Identification of PLXNA1-NRP2 bsAb with SEMA3F-mimicking mechanism and activities . A , schematic illustrating structural format of PLXNA1-NRP2 bsAbs. The bsAbs are based on a human IGG4 backbone, with the light chain and heavy chain of the anti-NRP2 half linked by a 34 amino acid GS flexible peptide linker. The anti-NRP2 moiety contains a knob mutation, while the anti-PLXNA1 moiety features a hole mutation. B , screening for bsAbs that induce dimerization of cell surface PLXNA1 and NRP2. Each table cell represents a unique PLXNA1-NRP2 bsAb. bsAbs inducing a dimerization ratio change greater than 1.5 are considered positive hits and highlighted in orange . C , PLXNA1-NRP2 bsAbs that significantly reduced p-AKT level. D , PLXNA1-NRP2 bsAbs that significantly reduced CCND1 expressing in qPCR assay. E , PLXNA1-NRP2 bsAbs that significantly inhibited proliferation of U-251 MG cells. All data are presented as the mean ± SEM from three experiments. aPLXNA1, anti-PLXNA1; bsAb, bispecific antibody; NRP, neuropilin; p-AKT, phosphorylation of AKT; PLXNA1, plexinA1; qPCR, quantitative PCR; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Mutagenesis, Expressing, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F

doi: 10.1016/j.jbc.2025.111056

Figure Lengend Snippet: Characterization of the binding mechanism of aPLXNA1-19-43 Fab to PLXNA1 . A , epitope mapping of aPLXNA1-19-43 Fab to PLXNA1. ELISA was used to evaluate binding of aPLXNA1-19-43 Fab to various PLXNA1 fragments. Results are indicated by ELISA readings, with positive (+) binding shown in red for readings >0.1 and negative (−) binding for readings <0.1. B , determination of binding affinity of aPLXNA1-19-43 Fab to PLXNA1 ECD and LBD. Representative sensorgrams from triplicate experiments are shown. The Fab was immobilized using biosensor tips coated with anti-mouse kappa antibody and subsequently exposed to a concentration series of PLXNA1-ECD or PLXNA1-LBD ( black and gray lines ). Data were fitted to a 1:1 binding model ( red lines ) to calculate binding constants. C , cryo-EM density map of the aPLXNA1-19-43 Fab in complex with PLXNA1-LBD. The 2:2 dimeric complex is displayed, with one subunit colored as follows: PLXNA1-LBD in blue , Fab heavy chain in cyan , and Fab light chain in red . D , schematic binding model of aPLXNA1-19-43 Fab with PLXNA1. The critical residues involved in binding are enlarged in the inserted panels . E , identification of critical residues on PLXNA1 that are involved in the binding of aPLXNA1-19-43 Fab. The Fab is shown to bind to both the SEMA and PSI domains of PLXNA1, with key interactions highlighted. F , binding model of P1943-Nb2cL to the PLXNA1–NRP2–SEMA3F complex. G , schematic representation of the binding mechanism between P1943-Nb2cL and the PLXNA1–NRP2–SEMA3F complex, providing a simplified visual overview. aPLXNA1, anti-PLXNA1; ECD, extracellular domain; Fab, fragment of antigen binding; LBD, ligand-binding domain; NRP, neuropilin; PLXNA1, plexinA1; PSI, plexin-semaphorin-integrin; SEMA, semaphorin.

Article Snippet: Dimerization between cell surface NRP2 and PLXNA1 proteins were determined using a luciferase complementation assay as described previously ( ). cDNAs of NRP2 and PLXNA1 were obtained from OriGene and R&D Systems respectively (NRP2 C220706, PLXNA1 RDC0967).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cryo-EM Sample Prep, Ligand Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , Retinal whole mounts from 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice were stained with anti-CD68 and anti-Iba-1 antibodies. Representative confocal images focused on the OPL are shown. The white arrowheads indicate CD68 + /GFP + or CD68 + /Iba-1 + microglia. b , c , Quantification of GFP + or Iba-1 + ( b ) and CD68 + /GFP + or CD68 + /Iba-1 + microglia ( c ) ( n = 4 mice per group). d Retinal sections from CX3CR1 GFP/GFP and C57BL/6J mice were stained with anti-GFAP and anti-Iba-1 antibodies. The yellow arrow indicates a resting microglial cell, and the white arrows indicate the dendritic migration of activated microglia into the ONL and INL. The yellow arrowhead indicates a resting astrocyte, and the white arrowheads indicate the extension of the dendritic processes of activated astrocytes into the IPL. e , Quantification of activated microglia in the ONL of retinas from CX3CR1 GFP/GFP and C57BL/6J mice ( n = 5–7 mice per group). f , qPCR analysis of proinflammatory molecules, including Tnfα , Il1b , Il6 , Cox2 and Nos2 , and anti-inflammatory cytokines, including Il10 and Il13 , in CX3CR1 GFP/GFP and C57BL/6J mouse retinas ( n = 6 mice per group). g , Retinal sections from CX3CR1 GFP/GFP and C57BL/6J mice were stained with anti-R/G opsin and anti-Iba-1 antibodies. Yellow arrowheads indicate cone photoreceptors. The boxed regions are highly magnified at the bottom, showing cone photoreceptors. Yellow arrows indicate resting microglia, and white arrowheads indicate the dendritic migration of activated microglia into the ONL. h , Quantification of cone photoreceptor cells in retinal sections ( n = 4–6 mice per group). i , j , Representative ERG images of 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice at 3 cd·s/m 2 under scotopic conditions ( i ) and at 10 cd·s/m 2 under photopic conditions ( j ). k , l , Amplitudes of ERG recordings under both scotopic and photopic conditions in 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice ( n = 11 mice per group). The data are presented as the mean ± s.e.m. and were analyzed via unpaired two-tailed Student’s t -tests (CX3CR1 GFP/GFP versus C57BL/6J, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). INL, inner nuclear layer. Scale bar, 20 µm.

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Staining, Migration, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , Volcano plot showing DEPs in retinas from 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice ( n = 5 mice per group). Red dots indicate upregulated proteins (FC >1.5), and green dots indicate downregulated proteins (FC <1/1.5), with a Q value <0.05. b – d , GO analysis of DEPs, shown separately for biological process ( b ), cellular component ( c ) and molecular function ( d ), between CX3CR1 GFP/GFP retinas and C57BL/6J retinas. e , KEGG pathway analysis of DEPs between CX3CR1 GFP/GFP retinas and C57BL/6J retinas. f , PPI analysis of DEPs. Pink dots indicate upregulated proteins, and blue dots represent downregulated proteins. g , KEGG network analysis of DEPs. The purple squares indicate the KEGG pathways, and a deeper color indicates greater significance. The red dots indicate upregulated DEPs, and the blue dots indicate downregulated DEPs. h , i , Western blotting analysis ( h ) and quantification of p-STAT3/STAT3 expression ( i ) in retinas from 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice ( n = 8–9 mice per group). j , Colocalization of p-STAT3 and Iba-1 in microglia from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age. The white arrowheads indicate p-STAT3 + /Iba-1 + microglia. Scale bar, 50 µm. k , Quantification of the percentages of p-STAT3 + /Iba-1 + microglia in j . l , qPCR analysis of Stat3 and proinflammatory molecule expression in microglia from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age. The results shown represent three to four independent experiments. The data are presented as the mean ± s.e.m. and were analyzed via unpaired two-tailed Student’s t -test (** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , qPCR analysis of Stat3 and proinflammatory molecule expression in retinas from 6-week-old CX3CR1 GFP/GFP mice treated with siSTAT3 or siCTR ( n = 5 mice per group). b , Retinal whole mounts from CX3CR1 GFP/GFP mice treated with siSTAT3 or siCTR were stained with an anti-CD68 antibody. The white arrowheads indicate CD68-positive microglia. c , Quantification of CD68 + /GFP + microglia ( n = 6–7 mice per group). d , Retinal sections from siSTAT3- or siCTR-treated CX3CR1 GFP/GFP mice were stained with an anti-GFAP antibody. The white arrows indicate the extension of the dendritic processes of activated astrocytes into the IPL, and the white arrowheads indicate the dendritic migration of activated microglia into the ONL. e , Quantification of microglial cell density in the ONL of retinas from CX3CR1 GFP/GFP mice treated with siSTAT3 or siCTR ( n = 5 mice per group). f , Quantification of the mean immunofluorescence intensity of GFAP in retinal sections ( n = 5 mice per group). g , Quantification of cone photoreceptors in retinas from siSTAT3- or siCTR-treated CX3CR1 GFP/GFP mice ( n = 5 mice per group). h , Retinal sections from siSTAT3- or siCTR-treated CX3CR1 GFP/GFP mice were stained with an anti-R/G opsin antibody. Yellow arrowheads indicate cone photoreceptors. The boxed regions are shown at higher magnification at the bottom. The white arrowheads show the dendritic migration of activated microglia into the ONL. i , Retinal sections from CX3CR1 GFP/GFP mice after treatment with siSTAT3 or siCTR were stained with anti-Caspase-3 and anti-PNA antibodies. The white arrowheads indicate Caspase-3 + /PNA + cells. j , k , Quantification of the number of PNA + cells ( j ) and percentages of Caspase-3 + /PNA + cells ( k ) in retinal sections ( n = 5 mice per group). l , Quantification of the mean immunofluorescence intensity of GFAP ( n = 5 mice per group). m , Retinal sections from CX3CR1 GFP/GFP mice treated with AAV-F4/80p-siSTAT3-mCherry or AAV-F4/80p-siCTR-mCherry were stained with an anti-GFAP antibody. The white arrowheads show the migration of activated microglia in the ONL, and the white arrows indicate the dendritic extension of activated astrocytes into the IPL from the NFL. n , Retinal sections from 6-week-old CX3CR1 GFP/GFP mice treated with AAV-F4/80p-siSTAT3-mCherry or AAV-F4/80p-siCTR-mCherry were stained with an anti-R/G opsin antibody. The white arrowheads indicate the migration of activated microglia in the ONL, and the yellow arrowheads indicate the R/G opsin + cone photoreceptors. o , Quantification of the number of R/G opsin + cone photoreceptors in n ( n = 5 mice per group). p , ERG recordings of CX3CR1 GFP/GFP mice at 6 weeks of age treated with AAV-F4/80p-siSTAT3-mCherry or AAV-F4/80p-siCTR-mCherry ( n = 10 mice per group). Scale bar, 20 µm. The data are presented as the mean ± s.e.m. and were analyzed via unpaired two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Expressing, Staining, Migration, Immunofluorescence, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a – d , Western blotting analysis ( a ) and quantification of CX3CR1 ( b ), p-STAT3 ( c ) and STAT3 expression ( d ) in BV2 cells transfected with siSTAT3 and siCX3CR1 or siCX3CR1 and siCTR. e , qPCR analysis of the expression of the proinflammatory molecules Cx3cr1 and Stat3 in BV2 cells after transfection with siSTAT3 and siCX3CR1 or siCX3CR1 and siCTR. f , qPCR analysis of proinflammatory cytokines in IMA2.1 cells treated with MCM-3, MCM-2 or MCM-1. g , qPCR analysis of A1- and A2-specific gene expression in IMA2.1 cells treated with MCM-3, MCM-2 or MCM-1. h – j , Flow cytometry analysis ( h ) and quantification of the percentages of Annexin-V + /PI − ( i ) and Annexin-V + /PI + ( j ) cells among 661W cells after treatment with MCM-3, MCM-2 or MCM-1. k – l , qPCR analysis of Caspase-3 ( k ) and Bax ( l ) expression in 661W cells treated with MCM-3, MCM-2 or MCM-1. The results shown represent three to five independent experiments. The data are presented as the mean ± s.e.m. and were analyzed via one-way ANOVA with Tukey’s multiple comparison test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Western Blot, Expressing, Transfection, Gene Expression, Flow Cytometry, Comparison

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , Flow cytometry analysis of p-STAT3 expression in microglia (CD11b + ), astrocytes (GFAP + ) and cone photoreceptors (R/G opsin + ) from the retinas of CX3CR1 GFP/GFP and CX3CR1 +/GFP or C57BL/6J mice at 6 weeks of age ( n = 3–4 mice per group). b , Quantification of the percentages of p-STAT3 + /GFP − , p-STAT3 + /GFP + and p-STAT3 − /GFP + cells in a . c , Quantification of the percentages of p-STAT3 + /GFAP − , p-STAT3 + /GFAP + and p-STAT3 − /GFAP + cells in a . d , Quantification of the percentages of p-STAT3 + /R/G-opsin − , p-STAT3 + /R/G opsin + and p-STAT3 − /R/G opsin + cells in a . e , Colocalization of p-STAT3 and GFAP in astrocytes from the retinas of 6-week-old CX3CR1 GFP/GFP or C57BL/6J mice. The white arrowheads indicate p-STAT3 + /GFAP + cells. Scale bar, 50 µm. f , Quantification of the percentages of p-STAT3 + /GFAP + cells in e . g , qPCR analysis of Stat3 and proinflammatory molecule expression in astrocytes from the retinas of 6-week-old CX3CR1 GFP/GFP or C57BL/6J mice. h , i , Western blotting analysis ( h ) and quantification of p-STAT3/STAT3 expression levels ( i ) in IMA2.1 cells treated with Colivelin TFA or PBS. j , qPCR analysis of proinflammatory molecules in IMA2.1 cells after Colivelin TFA or PBS treatment. k , qPCR analysis of A1- and A2-specific gene expression in IMA2.1 cells treated with Colivelin TFA or PBS. l ‒ o , Flow cytometry analysis ( l ) and quantification of the percentages of Annexin-V + /PI − ( m ), Annexin-V + /PI + ( n ) and Annexin-V − /PI + ( o ) cells among 661W cells treated with ACM-2 or ACM-1. The results shown represent three to four independent experiments. The data are presented as the mean ± s.e.m. and were analyzed via unpaired two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Flow Cytometry, Expressing, Western Blot, Gene Expression, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , UMAP plot showing eight unique microglial clusters from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age. b , Bubble chart showing marker gene expression in each cluster. c , Volcano plot showing DEGs in cluster aMG_1 ( Tnf dominant). The red dots indicate upregulated DEGs (FC >1.5), and the blue dots indicate downregulated DEGs (FC <1.5). d , PPI network of DEGs in cluster aMG_1 ( Tnf dominant). e , Violin plots showing Cx3cr1 , Tnf , Cd68 and Cxcl1 expression in each cluster. f , KEGG pathway analysis of DEGs in aMG_1 ( Tnf dominant). g , TNF signaling pathway in aMG_1 ( Tnf dominant) by GSEA. h , UMAP plots showing the AUC activities of TNF signaling in microglia from CX3CR1 GFP/GFP and C57BL/6J retinas. i, Colocalization of Iba-1 with CD68, TNF-α or CXCL1 in MACS-sorted microglia from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age. Scale bar, 20 µm. j , Activity of the top 20 TFs in 1,000 microglia from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age. k , Distribution of 8 different clusters among 1,000 microglia from CX3CR1 GFP/GFP and C57BL/6J retinas. l , Extended regulon activity of Stat3 among 1,000 microglia. m , Stat3 and Kdm6b expression in each cluster. n – r , Flow cytometry analysis ( n ) and quantification of CD11b-FITC + ( o ) and p-STAT3-APC + ( p ), TNF-α-Alexa Fluor-594 + ( q ) and p-STAT3-APC +/ TNF-α-Alexa Fluor-594 + ( r ) cells among CD11b + microglia from CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age ( n = 4 mice per group). The data are presented as the mean ± s.e.m. and were analyzed via unpaired two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). s , LR interaction network among eight different clusters. t , Circle plot showing TNF signaling networks. u , Heatmap showing the relative importance of each cluster on the basis of the computed four network centrality measures of TNF signaling. v , Pseudotime trajectory analysis of microglia.

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Marker, Gene Expression, Expressing, Activity Assay, Flow Cytometry, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , UMAP plot showing different retinal cell clusters from the retinas of 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice. b , Marker gene expression in each cluster. c , Pseudotime analysis of six distinct cone cell clusters. d , Expression of phototransduction-associated genes ( Gnat , Gnb1 , Nr2e3 , Nrl and Pdc ) and apoptosis-associated genes ( Egr1 , Fos , Gabrb3 , Gsdme and Prnp ) in cones over pseudotime. e , Inflammation-related gene expression in astrocytes from the retinas of 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice. f , Cell‒cell interactions between microglia and other retinal cells. g , Signaling changes in cones from the retinas of 6-week-old CX3CR1 GFP/GFP and C57BL/6J mice. h , LR interactions between microglia and cones. i , Violin plots showing CCL and CXCL signaling expression in different retinal cells. j , Expression of NF-ĸB signaling-associated DEGs in cones, bipolar cells, amines and RGCs between CX3CR1 GFP/GFP and C57BL/6J retinas. k , l , Flow cytometry analysis ( k ) and quantification ( l ) of the percentages of Annexin-V + /PI − , Annexin-V + /PI + and Annexin-V − /PI + cells among 661W cells pretreated with siAckr1 or siCTR and cocultured with pMCM-2 or pMCM-1. m , qPCR analysis of Ackr1 , Caspase-3 and Bax expression in 661W cells treated as described in k . n , o , Flow cytometry analysis ( n ) and quantification ( o ) of the percentages of Annexin-V + /PI − , Annexin-V + /PI + and Annexin-V − /PI + cells among 661W cells treated with CCL2 (100 ng/ml) after pretreatment with siAckr1 or siCTR. p , qPCR analysis of Ackr1 , Caspase-3 and Bax in 661W cells treated as described in n . The results represent three independent experiments. The data are presented as the mean ± s.e.m. and were analyzed via one-way ANOVA with Tukey’s multiple comparison test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Marker, Gene Expression, Expressing, Flow Cytometry, Comparison

Journal: Experimental & Molecular Medicine

Article Title: Microglial CX3CR1 deficiency regulates the selective vulnerability of cone photoreceptors via STAT3/CCL–ACKR1 signaling in the mouse retina

doi: 10.1038/s12276-025-01618-7

Figure Lengend Snippet: a , LR interactions between microglia and astrocytes. b , Upregulated signaling between microglia and astrocytes. c , Bmp signaling expression in different retinal cell clusters. d , Bmp2 expression in MACS-sorted microglia from CX3CR1 GFP/GFP and C57BL/6J mouse retinas. e , Bmp2 expression in BV2 cells following treatment with the recombinant CXCL1 protein (100 ng/ml) . f , qPCR analysis of Bmpr1a , Bmpr1b , Tnfα , Il1b and Il6 expression in IMA2.1 cells cocultured with cxMCM-2 or cxMCM-1 after pretreatment with siBmpr1a, siBmpr1b or siCTR. g , h , Western blotting analysis ( g ) and quantification ( h ) of p-STAT3 and STAT3 expression in IMA2.1 cells cocultured with cxMCM-2 or cxMCM-1 after pretreatment with siBmpr1a, siBmpr1b or siCTR. i , j , ELISA analysis of CXCL12 ( i ) and CCL2 ( j ) expression in cell supernatants from IMA2.1 cells cocultured with cxMCM-2 or cxMCM-1 after pretreatment with siBmpr1a, siBmpr1b or siCTR. The results shown represent three to four independent experiments. The data are presented as the mean ± s.e.m. and were analyzed via one-way ANOVA with Tukey’s multiple comparison test or unpaired two-tailed Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). k , Schematic illustration showing the role of CX3CR1/STAT3/CCL–ACKR1 signaling in mediating the selective vulnerability of cone photoreceptors in the mouse retina.

Article Snippet: For the detection of TNF-α, CCL2, CCL3, CCL4 and CXCL12 expressions in cell supernatants from MACS-sorted microglia or astrocytes in CX3CR1 GFP/GFP and C57BL/6J retinas at 6 weeks of age, TNF alpha Mouse Uncoated ELISA Kit (Invitrogen), MCP-1/CCL2 Mouse Uncoated ELISA Kit (Invitrogen), Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit (R&D system), Mouse CCL4/MIP-1 beta DuoSet ELISA (R&D system) and SDF-1 alpha/CXCL12 Mouse ELISA Kit (Invitrogen) were applied.

Techniques: Expressing, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test