Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3A and Nrp-1 in human lung tumour cell lines and CTL clone. a Sema-3A expression in human lung tumour cell lines. Total protein extracts were analysed by western blot using anti-Sema-3A mAb. The 16HBE cell line was included as a control. Full length and proteolytically processed proteins are indicated. b Co-expression of Nrp-1 and CD25, and Nrp-1 and PD-1 on P62 T cells stimulated with immobilised anti-CD3. c The P62 clone was unstimulated or stimulated with anti-CD3, pre-incubated with Sema-3A-Fc, and then labelled with anti-human IgG-Fc secondary mAb. Results are gMFI mean ± SEM of triplicate samples. d Sema-3A-Fc inhibits CTL migration toward a CXCL12 gradient. The T-cell clone was stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwell plates and exposed to a gradient of CXCL12 loaded in the lower chambers. The number of T cells that had migrated into the lower chambers was determined. Results are mean chemotaxis index ± SEM of triplicate samples. e Sema-3A-Fc inhibits CD8 + TIL migration toward CXCL12. CD8 + TIL isolated from three human NSCLC tumours were stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwells and exposed to a CXCL12 gradient. Results are mean chemotaxis index ± SEM of triplicates. f Cytotoxicity of the CTL clone toward autologous tumour cells. The P62 clone was stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward the cognate IGR-Pub cell line was determined. g Cytotoxic activity of freshly isolated TIL toward autologous tumour cells. TIL, freshly isolated from a NSCLC tumour, were stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward freshly isolated autologous tumour cells was determined. Data shown for cytotoxic assay correspond to one of three independent experiments. Means ± SEM two-tailed Student’s paired t test c , one-way ANOVA test with Bonferroni correction d , e or two-way ANOVA test with Bonferroni correction f , g . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Western Blot, Control, Incubation, Migration, Chemotaxis Assay, Isolation, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3B and Nrp-1 in B16F10 mouse melanoma model. a Expression of Sema-3B in B16F10 tumour cells. Total protein extracts from B16F10 cells cultured in vitro or isolated ex vivo from tumour grafts were analysed by western blot using anti-Sema-3B mAb. b Surface expression of Nrp-1 on CD4 + and CD8 + T cells infiltrating B16F10 melanoma engrafted in C57BL/6 mice. TIL from individual tumours were isolated at day 15 after tumour cell inoculation. T lymphocytes from spleens and TdLN of tumour-bearing mice were analysed in parallel. Percentages of positive cells are included. Right: percentages of Nrp-1 + cells among CD8 + and CD4 + T cells in TIL ( n = 29 and 31), splenocytes ( n = 29 and 31) and TdLN ( n = 11 and 16). c Expression of Nrp-1 and FoxP3 in CD4 + T cells. T lymphocytes from tumours ( n = 20), spleens ( n = 22) and TdLN ( n = 11) of B16F10 melanoma-bearing mice were analysed at day 15 by flow cytometry. Right: percentages of Nrp-1 among FoxP3 + and FoxP3 − CD4 + T lymphocytes from B16F10. d Expression of CD44 and CD62L on Nrp-1 + and Nrp-1 − CD8 + T cells from B16F10 TIL. Right: Distribution of naive, effector and memory T cells populations among Nrp-1 − and Nrp-1 + CD8 + TIL ( n = 10). Results are representative of 3–5 independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction b or two-way ANOVA test with Bonferroni correction c . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a , d

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Cell Culture, In Vitro, Isolation, Ex Vivo, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: The Nrp-1 + PD-1 hi TIL subset is enriched with activated antigen-specific CD8 + T cells. a Staining of CD8 + TIL with dextramers. C57BL/6 mice were engrafted with B16F10 and then vaccinated with Trp2 and gp100 peptides. On day 15, TIL were isolated from tumours. Right: Percentages of Trp2 ( n = 8) and gp100 ( n = 5) dextramer-positive T cells among Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + TIL. b Expression of IFNγ ( n =20) and TNF ( n =18) in Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + T cells. TIL were stimulated for 4 h with autologous tumour cells. c Degranulation of CD8 + TIL. TIL were stimulated with autologous tumour cells; then, T-cell subsets were analysed for expression of CD107a ( n = 18). d Cytotoxicity of freshly isolated CD8 + TIL. CD8 + TIL were pre-incubated in medium or with anti-Nrp-1, anti-PD-1 or a combination of both mAb; then, cytotoxicity toward autologous tumour cells was determined. e Increase in MHC-I and PD-L1 expression on tumour cells co-cultured with autologous CD8 + TIL. Kinetic studies of H-2-K b /-D b and PD-L1 expression on B16F10 co-cultured with CD8 + TIL. Expression profiles (left), percentages of positive cells (middle) and gMFI (right) of MHC-I (upper panels) and PD-L1 (lower panels) are shown. f Expression of perforin in CD8 + T cells. TIL were stimulated with autologous tumour cells in the absence or presence of neutralising anti-Nrp-1, anti-PD-1 or anti-Nrp-1 plus anti-PD-1 then, T-cell subsets were analysed for expression of perforin ( n = 5). g Anti-Nrp-1 re-establishes migration of Nrp-1 + PD-1 hi T cells toward B16F10. TIL were pre-incubated in medium or with neutralising anti-Nrp-1, -PD-1, a combination of both mAb or an isotype control. Cells were seeded in the upper chambers of transwells and then exposed to a gradient of B16F10 supernatant, enriched in Sema-3B, loaded in the lower chambers. The numbers of Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − T cells that had migrated were determined by flow cytometry. Results are representative of three independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction a – g . * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Staining, Isolation, Expressing, Incubation, Cell Culture, Migration, Control, Flow Cytometry

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a Quantitative real-time PCR (qPCR) analysis of Sema3b mRNA expression in sciatic nerves across postnatal development (P0, P7, P14, P28, and P60). n = 3 sciatic nerves per time point. b Transversal section of the P14 sciatic nerve showing that SEMA3B + SOX10 + MBP - SCs ensheath unmyelinated TUJ1 + axons. The arrowhead and arrow indicate SEMA3B + SOX10 + MBP - nmSCs and SEMA3B - SOX10 + MBP + mSCs, respectively. The experiment was repeated 3 times on 3 mice. c , d Representative TEM images of the sciatic nerve from P60 control and Sema3b ∆SC mice. nmSC lamellipodia are pseudo-colored to highlight their locations. Asterisks denote adjacent axons and circles denote partially wrapped axons. Arrowheads indicate lamellipodia protruding into the endoneurial space. The experiment was repeated 3 times on 3 mice. e , f Lateral views of 3D-reconstructed RB from the sciatic nerve of P60 control and Sema3b ∆SC mice. g Quantification of the RBM indexes from P60 control ( n = 7) and Sema3b ∆SC ( n = 7) mice. h Quantification of the RBM indexes from P7 ( n = 5 for both groups) and P28 Sema3b i∆SC ( n = 5 for both groups) mice with coil (CO) and tamoxifen (TM) treatment at P0. i Measurements of sensitivity to laser heat in control ( n = 6 for all groups) and Sema3b ∆SC ( n = 7 for all groups) mice. j Measurements of sensitivity to von Frey filaments in control ( n = 6) and Sema3b ∆SC ( n = 6) mice. All data are presented as mean ± SEM. * *p < 0.01, * p < 0.05, ns not significant. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a Schematic illustration of the strategy for generating Sema3b tgSC mice. b , c FISH on the sciatic nerve showing an increased Sema3b expression level in SOX10 + SCs of P14 Sema3b tgSC mice. Arrowheads and arrows in b indicate Sema3b + SOX10 + SCs and Sema3b - SOX10 + SCs. Note that in Sema3b tgSC mice, Sema3b expression is ubiquitous in all SOX10 + SCs, which concurrently express TOM. d Quantification of Sema3b transcript level with qPCR in the sciatic nerve of P14 control ( n = 6) and Sema3b tgSC ( n = 6) mice. e , f Representative TEM images of the sciatic nerve from P7 and P28 control and Sema3b tgSC mice. Asterisks and circles denote axons that are adjacent and partially wrapped, respectively. Arrowheads indicate lamellipodia protruding into the endoneurial space. g Quantification of the RBM indexes from P7 and P28 control ( n = 6 for both P7 and P28) and Sema3b tgSC ( n = 6 for both P7 and P28) mice. All data are presented as mean ± SEM. * p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP plot illustrating cell subtype diversity from P14 DRG. b Dot plot comparing expression levels of axon guidance pathway genes in Nefh − nmSNs and Nefh + mSNs. c UMAP plots illustrating the expression levels of Nefh , Nrp1 , Nrp2 , and L1cam in SNs. d–g Transverse sections of P14 sciatic nerve. NRP1 is expressed in TH + ( d ) and CGRP + ( e ) unmyelinated axons, and L1CAM is expressed in almost all unmyelinated axons ( f ). NRP1 and L1CAM are simultaneously expressed in subsets of unmyelinated axons ( g ). The experiment was repeated 3 times on 3 mice. h–k TEM images of the sciatic nerve from P60 control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN mice. l Quantification of the RBM indexes from control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN mice. n = 6 for all groups. m STED images illustrating the distribution of NRP1 and L1CAM particles within cultured sensory axon shafts following Fc and SEMA3B treatment. Arrowheads indicate particles with NRP1 and L1CAM co-localization. n Quantification of the number of L1CAM particles in the axon shaft with Fc ( n = 7 axon shafts from 3 DRGs) and SEMA3B ( n = 8 axon shafts from 3 DRGs) treatment. o Quantification of the numbers of particles with NRP1 and L1CAM co-localization in the axon shaft with Fc ( n = 7 axon shafts from 3 DRGs) and SEMA3B ( n = 8 axon shafts from 3 DRGs) treatment. p Representative images of the sensory axon shafts from control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN embryos showing the lateral filopodia of axon shaft. q Quantification of the percentages of collapsed sensory growth cones following Fc ( n = 5 DRGs from 3 control embryos) and SEMA3B (control, n = 6 DRGs/3 embryos; Nrp1 ∆SN , n = 6 DRGs/3 embryos; L1cam ∆SN , n = 5 DRGs/3 embryos; Nrp1/L1cam ∆SN , n = 6 DRGs/3 embryos) treatment. r Quantification of the filopodia numbers of cultured DRG shafts following treatment with Fc ( n = 7 axon shafts/3 DRGs) and SEMA3B (control, n = 5 axon shafts/3 DRGs; Nrp1 ∆SN n = 4 axon shafts/3 DRGs; L1cam ∆SN , n = 6 axon shafts/3 DRGs; Nrp1/L1cam ∆SN , n = 4 axon shafts/3 DRGs) treatment. All data are presented as mean ± SEM. *p < 0.05, ** p < 0.01. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Control, Cell Culture

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a STED images showing TUJ1 signals on axon bundles treated with Fc and SEMA3B. Arrowheads indicate axonal beading. b Fasciculation index is calculated as the axonal area divided by the total area. c Quantification of the fasciculation indexes for axon bundles treated with Fc and SEMA3B. n = 6 axon shafts from 3 DRGs for both groups. d KEGG pathway analysis of up-regulated DEGs in nmSN. Pathway enrichment was analyzed by a two-tailed Wald test with Benjamini-Hochberg correction (FDR-adjusted p-value < 0.05 and |log₂ fold change|> 0.25). e Dot plot illustrating genes in the cell adhesion molecules pathway. f , g STED images of P7 Plp1 CreERT2 ;R26 tdTom RBs. Dotted lines demarcate RB boundaries. Asterisks denote axons ensheathed by TOM + mSCs. The experiment was repeated 3 times on 3 mice. f’ , g’ Magnified views of the boxed areas in ( f ) and ( g ). Dotted lines demarcate unmyelinated axon boundaries. Arrowheads indicate lamellipodia undergoing separating fasciculated axons. The asterisk in ( g’ ) denotes an axon wrapped by SC lamellipodia, with strong CDH2 signals in the contact region. h STED images showing TUJ1 signals on axon bundles. i Quantification of the fasciculation indexes for axon bundles with SEMA3B or SEMA3B+Dynasore treatment. n = 6 axons bundles from 3 DRGs for both groups. j , k L1CAM and CDH2 particles on the membrane of sensory axon shafts from E14.5 DRG. l Quantification of artificial fluorescence intensity of L1CAM on the membrane of sensory axon shafts. n = 7 axon bundles from 3 DRGs for all groups. m Quantification of artificial fluorescence intensity of CDH2 on the membrane of sensory axon shafts. n = 6 axon bundles from 3 DRGs for all groups. n–p STED images of the RBs from P7 control, Sema3b tgSC , and Sema3b ∆SC mice. Dotted lines delineate the assumed RB boundaries. q Quantification of the numbers of L1CAM particles per RB. n = 6 RBs from 3 mice for all groups. r Schematic summary: nmSC-derived SEMA3B induces the endocytosis of axonal CAMs, leading to axonal segregation. All data are presented as mean ± SEM. ** p < 0.01, *p < 0.05. Detailed statistics are p rovided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Two Tailed Test, Membrane, Fluorescence, Control, Derivative Assay

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP plot illustrating L1cam expression in iSCs and nmSCs. b FISH on transverse section of P28 sciatic nerve. Arrowheads indicate L1cam + Scn7a + SOX10 + nmSCs and arrows indicate L1cam - Scn7a - SOX10 + mSCs. The experiment was repeated 3 times on 3 mice. c STED image of the RB from P28 Plp1 CreERT2 ;R26 tdTOM sciatic nerves. The experiment was repeated 3 times on 3 mice. c’ Magnified view of the boxed area in ( c ) illustrating a high L1CAM expression level in the contacting area between TUJ1 + axons and TOM + SC lamellipodia. Note the presence of L1CAM signals in TOM + SC lamellipodia. d Drawing a line to calculate the fluorescence intensity across the section. e Quantification of the fluorescence intensities of TOM, TUJ1, and L1CAM signals. f Representative E14.5 DRG SNs cultured on Fc- and L1CAM-coated coverslips. g Quantification of the numbers of attached sensory neurons. n = 6 independent experiments for both group. h SCs purified from P0 sciatic nerves cultured on Fc- or L1CAM-coated coverslips. i Quantification of the numbers of attached SCs. n = 6 independent experiments for both groups. j Assay for mimicking the acute and chronic effect of SEMA3B on attached SNs and SCs on L1CAM-coated coverslip. k Images showing attached SNs under different conditions. l Quantification of the numbers of attached SNs under different conditions. n = 6 independent experiments for all groups. m Images showing attached SCs under different conditions. n Quantification of the numbers of attached SCs under different conditions. n = 6 independent experiments for all groups. o Schematic representation of the three biological events involved in RB assembly: nmA-nmA adhesion, nmA-nmA deadhesion, and nmA-nmSC adhesion. The corresponding molecular mechanisms are depicted. nmSC-derived SEMA3B interacts with axonal NRP1/L1CAM receptor complex to shift CAM-induced nmA-nmA adhesion to CAM-induced nmA-nmSC adhesion. The lower panel presents STED images illustrating the dynamic distribution patterns of CAM during the three events of RB assembly. All data are presented as mean ± SEM. ** p < 0.01, *p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Fluorescence, Cell Culture, Purification, Derivative Assay

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP visualization of SCs from scRNA-seq data in sham mice and nerve-injury mice. b Relative proportions of SC lineages in total SCs of sham mice and nerve-injury mice. c Dot plot showing expression levels of Scn7a , Sema3b , and L1cam in SCs of sham mice and nerve-injury mice. d Cartoon depicting that the distal part of the injury site is harvested for analysis. e Sema3b expression levels in SOX10 + SC following different days post ligation (dpl) of control mice. f Sema3b expression levels in SOX10 + SCs following different days post-ligation (dpl) of Sema3b tgSC mice. Sema3b transcript levels demonstrated relatively stable after nerve injury. g Quantification using qPCR showing Sema3b transcript levels in sciatic nerves without injury (sham) and with 7 and 21 days post ligation (dpl). n = 6 mice for all groups. h–j Representative TEM images of the sciatic nerves from control and Sema3b tgSC mice. Note that the contact area between nmSC lamellipodia and axons is significantly decreased in the RB of control mice after injury. In contrast, the deficient RB assembly is noticeably reduced in Sema3b tgSC mice with injury. Asterisks and circles indicate axons that are adjacent and partially wrapped, respectively. Arrowheads indicate lamellipodia protruding into the endoneurial space. k Quantification of RBM indexes of control and Sema3b tgSC mice without and with nerve crush. n = 6 mice for all groups. l Measurements of sensitivity to von Frey filaments in control and Sema3b tgSC mice without and with nerve injury. n = 6 mice for all groups . m Measurements of sensitivity to hot laser in control and Sema3b tgSC mice without and with nerve injury. n = 6 mice for all groups. All data are presented as mean ± SEM. * *p < 0.01, *p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Ligation, Control

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Decreased Sema3B in perihematomal tissue is associated with neurological recovery after ICH. ( A ) Volcano plot from RNA-seq comparing perihematomal tissue following ICH with Sham controls, showing differentially expressed genes; Sema3b is markedly downregulated. Abbreviations: Sema3b , semaphorin 3B; Ccl4 , C–C motif chemokine ligand 4; Clec4d , C-type lectin domain family 4 member D; Tacstd2 , tumor-associated calcium signal transducer 2; Kcnk5 , potassium two pore domain channel subfamily K member 5; Ccdc153 , coiled-coil domain containing 153. ( B , C ) Immunoblotting for relative Sema3B protein expression in perihematomal tissue from 0 h (Sham) to 7 days after ICH ( n = 6). ( D ) Quantitative PCR assessment of relative Sema3B transcript abundance in perihematomal tissue from 0 h (Sham) to 7 days after ICH ( n = 6). ( E , F ) Representative immunoblots showing relative Sema3B protein levels in N2a cells stimulated with hemoglobin (Hb) ( n = 3). ( G ) Representative immunofluorescence images showing Sema3B expression in the perihematomal region at 72 h after ICH ( n = 3; scale bar = 1 mm). ( H ) Representative confocal immunofluorescence images showing neuronal Sema3B expression at 0 h (Sham) and at the 72 h time point after ICH ( n = 3; scale bar = 10 μm). ( I ) Correlation analysis revealed that higher Sema3B protein levels in the perihematomal tissue on day 3 after ICH were associated with a faster rate of neurological functional recovery during the first three days ( n =10). (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: RNA Sequencing, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Functional Assay

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B facilitates microglial clearance of the hematoma through PlexinA1 after ICH. ( A , B ) Protein immunoblot analysis of relative PlexinA1 protein expression in perihematomal tissue from 0 h (Sham) to 7 days after ICH ( n = 6). ( C ) qPCR measurement of relative PlexinA1 mRNA levels in perihematomal tissue from 0 h (Sham) to 7 days after ICH ( n = 6). ( D , E ) Western blot detection of relative PlexinA1 protein abundance in microglia stimulated with Hb ( n = 3). ( F ) Representative immunofluorescence images showing PlexinA1 expression in the perihematomal region at 72 h after ICH ( n = 3; scale bar = 1 mm). ( G ) Representative confocal immunofluorescence images showing PlexinA1 expression in microglia at 0 h (Sham) and on day 3 following ICH ( n = 3, scale bar = 10 μm). ( H ) Representative schematic of hematomas on day 3 after ICH ( n = 5). ( I ) Quantification of hematoma volume on day 3 after ICH in each group ( n = 5). ( J ) Analysis of residual hemoglobin content on day 3 after ICH ( n = 5). ( K ) Neurological deficit scores (NDS) on day 3 after ICH ( n = 5). ( L , M ) Representative flow-cytometry histograms and quantification of mean fluorescence intensity (MFI) for erythrophagocytosis in microglia under the indicated treatments ( n = 3). ( N ) Representative confocal immunofluorescence images of erythrophagocytosis in microglia under the indicated treatments ( n = 3, scale bar = 10 μm) (ns = no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Western Blot, Expressing, Quantitative Proteomics, Immunofluorescence, Flow Cytometry, Fluorescence

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B increases HO-1 and TREM2 expression in perihematomal brain tissue and microglia after ICH. ( A – C ) Immunoblotting showing relative HO-1 and TREM2 protein expression in microglia under the different treatments ( n = 4). ( D , E ) Quantitative PCR (qRT-PCR) assessment of HO-1 and TREM2 transcript levels in microglia under the different treatments ( n = 3). ( F – H ) Protein analysis by Western blot for HO-1 and TREM2 in perihematomal tissue from ICH mice on post-ICH day 3 under the different treatments ( n = 4) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B upregulates HO-1 and TREM2 expression via Nrf2. ( A , B ) Immunoblotting for relative Nrf2 protein expression in microglia under the different treatments ( n = 3). ( C ) Quantitative PCR (qRT-PCR) assessment of relative Nrf2 transcript levels in microglia under the different treatments ( n = 3). ( D ) Confocal micrographs depicting Nrf2 expression in microglia under the different treatments ( n = 3; scale bar = 10 μm). ( E – G ) Protein analysis by Western blot for HO-1 and TREM2 in microglia under the different treatments ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B enhances erythrophagocytosis by microglia via the TREM2/DAP12/SYK/PI3K/AKT pathway. ( A – F ) Immunoblotting for relative levels of DAP12, phospho-SYK, phospho-PI3K, phospho-AKT, and phospho-mTOR in microglia under the different treatments ( n = 3). ( G , H ) Representative flow-cytometry histograms and quantitative analysis of the MFI for erythrophagocytosis in microglia under the different treatments ( n = 3). ( I ) Confocal micrographs depicting erythrophagocytosis in microglia under the different treatments ( n = 3, scale bar = 10 μm) (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Western Blot, Flow Cytometry

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Interaction between PlexinA1 and TREM2 in microglia is enhanced by Sema3B, synergistically promoting hematoma phagocytosis. ( A ) The interaction network between PlexinA1 and TREM2 was determined using the STRING database ( https://string-db.org (accessed on 2 June 2025)) and subsequently plotted with Cytoscape software (v3.10). ( B ) Detection of the PlexinA1–TREM2 interaction in microglia under basal conditions by co-immunoprecipitation and immunoblotting. ( C , D ) Co-immunoprecipitation and immunoblotting analyses of the PlexinA1–TREM2 interaction in microglia under the different treatments, with quantification (n = 3) (** p < 0.01).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Software, Immunoprecipitation, Western Blot

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B promotes microglial polarization toward the M2 phenotype and ameliorates microglia-mediated inflammation after ICH. ( A – D ) Immunoblotting for relative protein expression of the M1 marker CD86 and the M2 marker CD206 in microglia under the different treatments ( n = 3). ( E – H ) Protein analysis by Western blot for phosphorylated NF-κB and IκB-α in microglia under the different treatments ( n = 3). ( I – K ) Quantitative PCR assessment of IL-1β, TNF-α, and IL-6 transcript levels in microglia under the different treatments ( n = 3). ( L – N ) ELISA measurement of IL-1β, TNF-α, and IL-6 protein concentration in perihematomal tissue on post-ICH day 3 under the different treatments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Western Blot, Expressing, Marker, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Antioxidants

Article Title: Neuron-Derived Sema3B Facilitates Microglial Hematoma Clearance After Intracerebral Hemorrhage

doi: 10.3390/antiox15020220

Figure Lengend Snippet: Sema3B promotes hematoma absorption after ICH by enhancing PlexinA1-mediated microglial phagocytic function. Exogenous supplementation of Sema3B binds to its receptor PlexinA1, activating the DAP12-dependent signaling pathway (Syk-PI3K-AKT-mTOR) and NRF2 in microglia, thereby increasing the expression of TREM2 and HO-1 to facilitate microglia-mediated hematoma clearance, while suppressing neuroinflammation by inhibiting the NF-κB pathway and reducing pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). This figure was drawn by Figdraw.

Article Snippet: Recombinant mouse Sema3B protein (R&D Systems, Minneapolis, MN, USA) was administered via intracerebroventricular injection at the following coordinates: AP −0.3 mm, ML +1.0 mm, DV −2.5 mm.

Techniques: Expressing