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

Journal: Journal of Cell Science

Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

doi: 10.1242/jcs.232645

Figure Lengend Snippet: VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and cDNA constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.

Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

Techniques: Phospho-proteomics, Activation Assay, Transfection, Small Interfering RNA, Construct, Western Blot, One-tailed Test

Journal: Journal of Cell Science

Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

doi: 10.1242/jcs.232645

Figure Lengend Snippet: CXCR4 is required for VLA-4-dependent cell migration in a ligand-independent manner. (A) CAGHPSE, HMEC-1 or M14 cells were transfected with two different CXCR4 siRNAs for 48 h prior to 16 h transfilter migration assays towards 100 µg/ml IIICS. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells. (B) CAGHPSE, HMEC-1 or M14 cells transfected with CXCR4 siRNA for 48 h were analyzed by immunoblotting for integrin α4-pS988, PKA pT197, CXCR4 or β-actin. (C) 16 h transfilter migration assays towards IIICS were performed with CAGHPSE and M14 cells treated with or without 10 µg/ml CXCR4-blocking antibody or 10 µM AMD3100 in the absence or presence of 20 ng/ml SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments. (D) CAGHPSE or M14 cells were transfected with or without VEGFR2 siRNA for 48 h. 16 h transfilter migration assays towards 100 µg/ml IIICS were performed with or without 30 µM SSTNVEGFR2 or 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (E) HMEC-1 cells were plated on 100 µg/ml IIICS with 30 µM SSTNVEGFR2 for 2.5 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2 antibody. The VEGFR2-associated complexes were analyzed by immunoblotting for VEGFR2 (with mouse anti-VEGFR2), CXCR4, AC7, PKA, hSdc1 or α4 integrin. (F) HMEC-1 or M14 cells were transfected with two different Sdc1 siRNA for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2. The associated complexes were probed with mouse anti-VEGFR2, anti-CXCR4, anti-α4 integrin, anti-AC7, anti-hSdc1 or anti-PKA antibodies. Silencing of Sdc1 expression was confirmed by immunoprecipitation with rabbit polyclonal anti-Sdc1 and probed with mouse anti-human Sdc1 antibody. (G) CAGHPSE or M14 cells were transfected with VEGFR2 siRNAs for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit polyclonal anti-Sdc1 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, CXCR4, Sdc1 or α4 integrin. The whole-cell lysates were analyzed by immunoblotting for VEGFR2. (H) HMEC-1 cells were transfected with two different CXCR4 siRNAs for 48 h and then cell lysates were subjected to immunoprecipitation with anti-VEGFR2 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, VEGFR2 or Gβγ. The whole-cell lysates were analyzed by immunoblotting for CXCR4.

Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

Techniques: Migration, Transfection, One-tailed Test, Western Blot, Blocking Assay, Immunoprecipitation, Expressing

Journal: Journal of Cell Science

Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

doi: 10.1242/jcs.232645

Figure Lengend Snippet: VEGFR2 activates CXCR4 by phosphorylating Y135 in its DRY motif. (A) CAGHPSE, HMEC-1 or M14 cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM vandetanib or 30 µM SSTNVEGFR2 for 2.5 h. CXCR4 was immunoprecipitated with rabbit anti-CXCR4 antibody and the CXCR4-containing complexes were analyzed by immunoblotting with anti-phosphotyrosine or rat anti-CXCR4 antibody. (B–F) CAGHPSE, HMEC-1 or M14 cells were co-transfected with CXCR4 3′UTR siRNA and CXCR4 cDNA lacking the 3′UTR (GFP-tagged WT and Y→F mutants or GFP alone) for 48 h. The cells were then analyzed by flow cytometry, immunoprecipitation and migration assays. (B) GFP-tagged WT or Y135F CXCR4 mutant HMEC-1 cells were incubated with PBS alone or 200 nM SDF-1 in PBS at 4°C. After incubation with 10 µg/ml anti-SDF-1 antibody versus an isotype-matched control mouse IgG1 for 1 h, cells were fixed and blocked with 3% BSA in PBS. The bound antibody was detected with a RPE-labeled anti-mouse IgG and analyzed by flow cytometry for SDF-1-bound CXCR4. (C) CAGHPSE cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-VEGFR2, anti-α4 integrin, anti-hSdc1, anti-AC7, anti-PKA or anti-CXCR4 antibodies. (D) M14 cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-phosphotyrosine (pY20) or anti-CXCR4 antibodies. (E) Quantification of 16 h transfilter migration of CAGHPSE cells towards 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 is plotted as the mean±s.d. of three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (F) CAGHPSE cells were kept in suspension or plated on 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 with or without 10 µM vandetanib for 2 h. Cell lysates were subjected to immunoprecipitation with anti-GFP or anti-GTP-Gαi antibodies, then probed with anti-Gαi antibody. The whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988 and β-actin.

Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

Techniques: Immunoprecipitation, Western Blot, Transfection, Flow Cytometry, Migration, Mutagenesis, Incubation, Control, Labeling, One-tailed Test, Suspension

Journal: Journal of Cell Science

Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

doi: 10.1242/jcs.232645

Figure Lengend Snippet: The CXCR4 Y135D phosphomimetic mutant rescues the effects of VEGFR2 inhibition. (A,B) CAGHPSE, HMEC-1 or M14 cells were transfected with GFP alone, GFP–CXCR4 WT, or GFP–CXCR4 Y135D together with siRNA targeting 3′UTR of endogenous CXCR4 for 48 h. (A) 16 h transfilter cell migration assay towards 100 µg/ml IIICS was performed in the absence or presence of 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01, n.s., not significant. (B) M14 cell lysates were subjected to immunoprecipitation with anti-GTP-Gαi and probed for Gαi. Whole-cell lysates were analyzed by immunoblotting for Gαi or α4 integrin (total or pS988). (C) Model depicting how paxillin bound to VLA-4 causes inherent inhibition of Rac GTPase at this site due to its binding of ArfGAP (Nishiya et al., 2005). VEGFR2 forms a complex with CXCR4, AC7 and PKA, but this complex is inactive unless Sdc1 is shed. Trimming of the HS chains on Sdc1 by HPSE facilitates its shedding by MMP9 (Purushothaman et al., 2010; Yang et al., 2007). Sdc1 freed of its membrane anchorage couples VEGFR2 and its integrin phosphorylation machinery to VLA-4 clusters. VEGFR2 activated by this clustering event phosphorylates CXCR4 at Y135, activating its heterotrimeric G-protein. Gβγ freed of Gαi activates AC7, generating cAMP that activates PKA. PKA phosphorylation of the α4 integrin cytoplasmic domain at S988 dissociates paxillin–ArfGAP from VLA-4, allowing local lamellipodium formation and directed cell migration.

Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

Techniques: Mutagenesis, Inhibition, Transfection, Cell Migration Assay, One-tailed Test, Immunoprecipitation, Western Blot, Binding Assay, Membrane, Phospho-proteomics, Migration

Journal: Journal of Cell Science

Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

doi: 10.1242/jcs.232645

Figure Lengend Snippet: sSdc1-mediated VEGFR2 activation suppresses cytotoxic T cell and NK cell migration by displacing VLA-4 from LFA-1. (A) Lysates from CAGHPSE cells, NK cells and T cells were analyzed by immunoblotting for VEGFR2, VLA-4, LFA-1, Sdc1, PKA, CXCR4, AC7 and β-actin (loading control). (B) NK cells and T cells were plated on 10 µg/ml VCAM-1 in the absence or presence of 30 µM SSTNVEGFR2 for 2.5 h. VEGFR2 was immunoprecipitated and the VEGFR2-containing complexes analyzed by immunoblotting for VEGFR2, AC7, Sdc1, PKA, CXCR4, α4 integrin [total (VLA-4) and pS988], LFA-1 and VLA-5. (C) NK cells and T cells were plated on either ICAM-1 or VCAM-1 in the absence or presence of SDF-1 with or without anti-VLA-4 or anti-LFA-1 antibodies or CXCR4 inhibitor (AMD3100). Attached NK cells and T cells in five random images for each experiment were quantified and graphed as a percent of cells treated with SDF-1 alone (set to 100%). All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (D) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone, 10 µg/ml ICAM-1 alone, or a mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1, analysis after treatment with 30 µM SSTNVEGFR2, 10 µg/ml anti-VLA-4, or 10 µg/ml anti-LFA-1 in the absence or presence of SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells on each ligand; **P<0.01, n.s., not significant between treatments. (E) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 plus 10 µg/ml VCAM-1 was performed as in D in the absence or presence of 30 µM SSTNVEGFR2, 1 µM vandetanib or 10 µM AMD3100. All data were compared using the unpaired one-tailed t-test. **P<0.01, n.s., not significant. (F) NK cells and T cells were plated on 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1 in the absence or presence of SSTNVEGFR2. Cell lysates were subjected to immunoprecipitation of VLA-4 and probed for VLA-4 or LFA-1. Whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988, VEGFR2-pY1054/1059 or β-actin. (G) Model depicting how LFA-1 and VLA-4 on NK cells and T cells associate in an adhesion complex in which signaling from FAK and PYK2, localized to VLA-4 by paxillin, transregulates LFA-1 activity leading to LFA-1-dependent cell migration (Cantor et al., 2015; Rose et al., 2003). If Sdc1 is shed by the leukocytes, or high levels of shed Sdc1 accumulate in the tumor microenvironment from HPSE-overexpressing tumor cells, the shed syndecan couples VEGFR2 and its phosphorylation machinery to VLA-4. Activation of CXCR4 by SDF-1, or by VEGFR2-mediated phosphorylation of Y135 in the CXCR4 cytoplasmic loop 2, causes phosphorylation of S988 in the α4 integrin subunit, displacing paxillin and suppressing LFA-1-mediated influx of cytotoxic leukocytes to the tumor. SSTNVEGFR2, which reverses the effects of shed syndecan, relieves this immunosuppression while at the same time blocking tumor cell migration and metastasis (see Fig. 6C).

Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

Techniques: Activation Assay, Migration, Western Blot, Control, Immunoprecipitation, One-tailed Test, Activity Assay, Phospho-proteomics, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

doi: 10.1074/jbc.m110.146050

Figure Lengend Snippet: FIGURE 3. Engineering a full-length PHR1 cDNA. Indicated by thin black lines are products generated by 5-RACE, 3-RACE, and RT-PCR that were used to form the full-length PHR1 cDNA. An N-terminal cassette was formed by joining products A and B using a NotI site. A C-terminal cassette was formed by joining products G, H, and F using SphI and EagI. The N- and C-terminal cassettes were joined to form the full-length PHR1 cDNA using a unique (*) SmaI site.

Article Snippet: 3 -RACE—Poly(A)RNA fromChlamydomonas strain cc-125 was reverse-transcribed using cDNA cloning primer (Integrated DNA Technologies) and ImProm-II reverse transcriptase (Promega).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction