Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 1. Expression Profiling Identifies Endothelial Cell-Specific Secreted Sema3G as a Potential Promoter of Communications from the Vasculature to Neurons (A) Venn plot representing the number of intersecting secretome genes which are preferentially expressed in ECs over other cell types (FPKM fold change > 20). ECs: endothelial cells, Ast: astrocytes, MG: microglia, Neu: neurons, OPC: oligodendrocyte precursor cells. (B) Heatmap of fold changes (log2) in expression of 18 candidate genes extracted from 2 databases that are highly and specifically expressed in brain ECs. (C) RNA-seq data (Zhang et al., 2014) showed that Sema3G is expressed mainly in endothelial cell rather than other cell type in brain. FPKM: Fragments per kilobase of transcript per million mapped reads. (D) Immunoblots of equal volumes of human cerebrospinal fluid revealed a pronounced decrease of Sema3G in Alzheimer’s disease patients (AD) compared with control (Con). (E) Immunoblots of Sema3G protein level in primary BMVECs lysates of WT mice.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Expressing, RNA Sequencing, Western Blot, Control

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 3. Loss of Sema3G in ECs Decreases the Synaptic Strength of the CA3-to-CA1 Neural Circuits (A) Representative traces of AMPAR- and NMDAR-mediated responses in CA1 pyramidal neurons of the indicated genotypes; filled circles indicate the time points at which measurements were taken (left). Quantification of the NMDAR:AMPAR ratio (right, p < 0.05, Student’s t test). n: the number of cells from 9 or 10 mice. (B) Representative evoked NMDAR current recorded in CA1 pyramidal neurons by electrical stimulation of the Schaffer collaterals in the indicated groups (left). Summary data are shown in right (p < 0.001, two-way ANOVA). n: the number of cells from 4 or 5 mice. (C) Representative evoked NMDAR current recorded in CA1 pyramidal neurons (left, black for Sema3Gf/f and red for Cdh5-Cre;Sema3Gf/f). Summarized decay time constant of isolated NMDAR EPSCs (right, p > 0.05, Student’s t test). n: the number of cells from 4 or 5 mice. (D) Representative evoked AMPAR current recorded in CA1 pyramidal neurons by electrical stimulation of the Schaffer collaterals (left). Summary data are shown in right (p < 0.05, two-way ANOVA). n: the number of cells from 5 mice. (E) AAV vector used to express hChR2 (H134R)-mCherry under the CaMKIIa promoter. (F) ChR2-mCherry is expressed in the CA3 region (top, scale bar, 500 mm). Co-localization of ChR2-mCherry (red) and CaMKIIa (green) in the CA3 (bottom, scale bar, 20 mm). Higher magnification images inserted show expression of ChR2 in CA3 pyramidal neurons. (G) A putative pyramidal neuron was recorded using a biocytin-containing solution and co-labeled with CaMKIIa (Gray). Schaffer collateral axons express ChR2- mCherry (red, scale bar, 20 mm in the left and 5 mm in the right). (H) Schematic of the recording configuration. Patch-clamp recording of a CA1 pyramidal neuron and optogenetic stimulation of ChR2-mCherry-positive axons projecting from CA3 neurons. (I) Examples of blue light-evoked CA3-CA1 projection EPSCs recorded in CA1 pyramidal cells of the indicated genotypes (left). Summarized EPSC amplitude (right, p < 0.001, Student’s t test). n: the number of cells from 5 or 6 mice. Data are presented as mean ± SEM; *p < 0.05, ***p < 0.001; ns, not significant, p > 0.05. See also Figure S4 and Table S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Isolation, Plasmid Preparation, Expressing, Labeling, Patch Clamp

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 4. Loss of Sema3G Decreases mEPSC Frequency and Dendritic Spine Density in CA1 Pyramidal Neurons (A–C) Representative mEPSCs traces (top) and the mEPSC frequencies (bottom) recorded in CA1 pyramidal neurons of the indicated groups (A, p < 0.001; B, p < 0.01; C, p < 0.001). n: the number of cells from 6 mice in (A), 5 mice in (B), and 3 mice in (C). (D) z stack images of dendritic segments in the apical dendrites of CA1 pyramidal neurons of the indicated genotypes (scale bar, 5 mm). (E–H) Quantification of the spine density: total spine density (E, p < 0.05), mushroom spine density (F, p < 0.01), stubby spine density (G, p > 0.05), and thin spine density (H, p > 0.05). n: the number of dendrites from 5 mice. (I) Schematic representation of AAV-BR1 constructs indicating the inverted terminal repeats (ITR) at both ends, a CAG promoter-driven EGFP (BR1-Con), or the CAG promoter-driven iCre linked by the 2A peptide to EGFP (BR1-iCre). (J) Representative z stack image from brain cortex, three weeks after vector injection. BR1-Con-transduced cells (green) were positive for the endothelial marker lectin (red). Scale bars, 100 mm. (K) Relative mRNA levels of Sema3G in brain ECs of Sema3Gf/f mice injected with BR1-Con or BR1-iCre (p < 0.01). n = 3 mice.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Construct, Plasmid Preparation, Injection, Marker

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 5. Sema3G Increases Excitatory Synapse Density and Excitatory Synaptic Transmission via Nrp2 (A) Immunofluorescence staining of Nrp2 (red, i) and CaMKIIa (green, ii) in CA1 (scale bar, 50 mm). Higher magnification is depicted as a z stack image (iv, scale bar, 15 mm). DAPI counterstaining (blue) indicates nuclei. (B) Alkaline phosphatase (AP)-Sema3G fusion protein was bound to brain sections of P5 (ii), P7 (iii), P20 (iv), and adult (v) WT mice to detect the endogenous Sema3G binding receptor (Nrp2 protein) distribution using a colorimetric AP activity assay. AP-incubated P5 brain slice was set as control (i). Scale bar, 300 mm. (C) Relative mRNA level of Nrp2 in 4-day primary cultures of rat dissociated neurons transfected with Con-RNAi or Nrp2-RNAi at DIV 0 (p < 0.001, Student’s t test). n = 3 independent experiments. (D) Representative confocal images of dendrites from DIV 14 primary hippocampal neurons of indicated groups. Dendrites were immunostained for vGluT1 (green) and PSD95 (red), with the merged images shown in yellow. Scale bar, 5 mm. (E–G) Quantification of the average density of vGluT1 puncta (E, Con-RNAi, p < 0.001; Nrp2-RNAi, p > 0.05; one-way ANOVA), PSD95 puncta (F, Con-RNAi, p < 0.001; Nrp2-RNAi, p > 0.05; one-way ANOVA), and vGluT1/PSD95 colocalized puncta (G, Con-RNAi, p < 0.001; Nrp2-RNAi, p > 0.05; one-way ANOVA) in neurons with the indicated treatment. n: the number of dendrites from 3 independent experiments. (H) Schematics of AAV constructs engineered to silence Nrp2 (Nrp2-RNAi, lower) or control (Con-RNAi, upper) by mir30 shRNA under the CaMKIIa promoter. (I) Top: RNAscope for Nrp2 probes (green) in CA1 stratum pyramidale in the indicated groups. DAPI counterstaining (blue) indicates nuclei (scale bar, 50 mm). Bottom: quantification of relative Nrp2 mRNA level (p < 0.001, Student’s t test). n = 3 mice. (J) EGFP expression (green) in the hippocampus 3 weeks after AAV-Nrp2-RNAi injection into CA1 region (scale bar, 500 mm). (K and L) Representative mEPSCs traces (top) and frequencies (bottom) recorded in CA1 pyramidal neurons from mice of the indicated groups injected with AAV in CA1 region (K, p < 0.01; L, p > 0.05; Student’s t test). n: the number of cells from 5 mice. Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001; ns, not significant, p > 0.05. See also Table S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Transmission Assay, Staining, Binding Assay, Activity Assay, Incubation, Slice Preparation, Control, Transfection, Construct, shRNA, RNAscope, Expressing, Injection

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 6. Sema3G Increases Excitatory Synapse Density and Excitatory Synaptic Transmission through Postsynaptic PlexinA4 Signaling (A) Representative confocal images of dendrites from DIV 14 primary hippocampal neurons of indicated groups. Dendrites were immunostained for vGluT1 (green) and PSD95 (red), with the merged images shown in yellow. Scale bar, 5 mm. (B–D) Quantification of the average density of vGluT1 puncta (B, Con-RNAi, p < 0.05; PlexinA4-RNAi, p > 0.05; one-way ANOVA), PSD95 puncta (C, Con-RNAi, p < 0.001; PlexinA4-RNAi, p > 0.05; one-way ANOVA), and vGluT1/PSD95 colocalized puncta (D, Con-RNAi, p < 0.001; PlexinA4-RNAi, p > 0.05; one-way ANOVA) in neurons with the indicated treatment. n: the number of dendrites from 3 independent experiments. (E) Representative mEPSC traces from primary hippocampal neurons of the indicated group. (F) Quantification of the normalized mEPSC frequency (Con-RNAi, p < 0.01; PlexinA4-RNAi, p > 0.05; one-way ANOVA). n = 16 or 17 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Transmission Assay

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 7. Rac1 Activation Is Required for Sema3G-Induced Increase in Dendritic Synapse Density (A) Immunostaining of vGluT1 (green) and PSD95 (red) in dendritic segments of primary hippocampal neurons showing the effects of Rac1 inhibitor NSC23766 (10 mM) and RhoA inhibitor Y-27632 (10 mM) treatment on Sema3G induced increases in synapse density (scale bar: 5 mm). (B–D) Quantification of the average density of vGluT1 puncta (B, vehicle treatment, p < 0.001; NSC23766 treatment, p > 0.05; Y-27632 treatment, p < 0.01; one- way ANOVA), PSD95 puncta (C, vehicle treatment, p < 0.001; NSC23766 treatment, p > 0.05; Y-27632 treatment, p < 0.001; one-way ANOVA), and vGluT1/ PSD95 colocalized puncta (D, vehicle treatment, p < 0.001; NSC23766 treatment, p > 0.05; Y-27632 treatment, p < 0.001; one-way ANOVA) in neurons with the indicated treatment. n: the number of dendrites from 3 independent experiments. (E) Representative mEPSC traces from primary hippocampal neurons of the indicated group. (F) Quantification of the normalized mEPSC frequency (vehicle treatment, p < 0.01; NSC23766 treatment, p > 0.05; one-way ANOVA). n = 16-17 cells. (G) Pull-down assay showing the effect of Sema3G on Rac1 activity (left). Quantification of Rac1 GTP fold change (right, p < 0.001, Student’s t test). n = 3 in- dependent experiments. (H) Immunostaining of vGluT1 (green) and PSD95 (red) in dendritic segments of primary hippocampal neurons transfected with GFP, GFP-WT-Rac1 or GFP-DN- Rac1 (dominant negative) with Sema3G or control treatment. Scale Bar: 5 mm. (I) Quantification of the average density of vGluT1/PSD95 colocalized puncta (GFP transfected, p < 0.001; GFP-WT-Rac1 transfected, p < 0.001; GFP-DN-Rac1 transfected, p > 0.05; one-way ANOVA) in neurons with the indicated treatment. n: the number of dendrites from 3 independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Activation Assay, Immunostaining, Pull Down Assay, Activity Assay, Transfection, Dominant Negative Mutation, Control

Journal: Neuron

Article Title: Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4.

doi: 10.1016/j.neuron.2018.12.036

Figure Lengend Snippet: Figure 8. Overexpressing Sema3G in the CA1 Restores Spine Density and Cognitive Function in Cdh5-Cre;Sema3Gf/f Mice (A) Schematics of AAV constructs engineered to overexpress ZsGreen (AAV-Con) or Sema3G (AAV-Sema3G) under the CMV promoter. (B) Immunoblots showing overexpression of Sema3G in hippocampus of AAV-Sema3G injected mice compared with AAV-Con injected mice (left). Quantification of normalized Sema3G level (right, Sema3Gf/f + AAV-Con versus Sema3Gf/f + AAV-Sema3G, p < 0.01; Cdh5-Cre;Sema3Gf/f + AAV-Con versus Cdh5-Cre; Sema3Gf/f + AAV-Sema3G, p < 0.01). n: the number of mice. (C) Experimental paradigm for behavior tests and spine morphology analysis. (D) Changes in contextual freezing during contextual memory retrieval in a contextual fear-conditioning test in the indicated groups (Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Con, p < 0.05; Cdh5-Cre;Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Sema3G, p < 0.001). n: the number of mice. (E) Changes in the spontaneous alternation behavior in the Y-maze test in the indicated groups (Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Con, p < 0.05; Cdh5-Cre;Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Sema3G, p < 0.01). n: the number of mice. (F) Representative confocal images of dendritic segments in the apical dendrites of CA1 pyramidal neurons of Sema3Gf/f and Cdh5-Cre;Sema3Gf/f mice with AAV-Con or AAV-Sema3G injection. Scale bar, 5 mm. (G–J) Quantification of the total spine density or the density of different subtypes: total spine density (G, Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV- Con, p < 0.05; Cdh5-Cre;Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Sema3G, p < 0.01), mushroom spine density (H, Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Con, p < 0.01; Cdh5-Cre;Sema3Gf/f + AAV-Con versus Cdh5-Cre;Sema3Gf/f + AAV-Sema3G, p < 0.01), stubby spine density (I, four groups, p > 0.05), and thin spine density (J, four groups, p > 0.05). n: the number of dendrites from 3 mice. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01; ##p < 0.01, ###p < 0.001, ns, not significant, p > 0.05, using one-way ANOVA followed by Tukey’s multiple comparisons test. See also Table S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (Control) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAGmir30shRNA (Nrp2) Obio Technology, Shanghai, China N/A AAV-CaMKIIa-EGFP-3 3 FLAG-mir30shRNA (PlexinA4) Obio Technology, Shanghai, China N/A AAV-CMV-Sema3G-3 3 Myc Hanbio Biotechnology, Shanghai, China N/A AAV-CMV-ZsGreen Hanbio Biotechnology, Shanghai, China N/A AAV-BR1-CAG-iCre-2A-EGFP Körbelin et al., 2016 N/A AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A Biological Samples Human cerebrospinal fluid samples China Brain Bank of Zhejiang University N/A Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Scientific Cat# D1306 Recombinant Sema3G protein This paper N/A Basic fibroblast growth factor (bFGF) R&D Systems Cat# 3139-FB-025 Picrotoxin Tocris Cat# 1128 CNQX Tocris Cat# 0190 DL-AP5 Tocris Cat# 0105 QX 314 bromide Tocris Cat# 1014 Tetrodotoxin (TTX) Tocris Cat# 1078 Biocytin Sigma Aldrich Cat# B4261 Spermine Sigma Aldrich Cat# S4264 NBT Roche Cat# 11383213001 BCIP Roche Cat# 11383221001 NSC23766 Selleck Chemicals Cat# S8031 Y-27632 Selleck Chemicals Cat# S1049 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Critical Commercial Assays Active Rac1 Pull-Down and Detection Kit Thermo Fisher Scientific Cat# 16118 EZ-ECL Chemiluminescence Detection Kit Biological Industries Cat# 20-500-120 HisTrap HP column GE Health Cat# 17-5248-01 TSA plus Fluorescence Kit PerkinElmer Cat# NEL741001KT Lucifer yellow CH Lithium Salt Invitrogen Cat# L453 Vectashield Mounting Medium Vector Laboratories Cat# H-1000 Nanogold-labeled goat anti-rabbit IgG Nanoprobes Cat# 2003 HQ silver enhancement kit Nanoprobes Cat# 2012 Protein-G coupled Sepharose beads GE Healthcare Cat# 17-0618-01 Sema3G ELISA kit Cusabio, Wuhan, China Cat# CSB-EL020986MO Rat Neuron Nucleofector Solution Lonza Cat# VPG-1003 PrimeScript RT Master Mix Takara Cat. #RR036A SYBR Premix Ex Taq Takara Cat. #RR820A RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat# 323100 Plxna4-C2 probe Advanced Cell Diagnostics Cat# 515491-C2 Nrp2-C1 probe Advanced Cell Diagnostics Cat# 500661 RNAscope 3-plex Positive control probe Advanced Cell Diagnostics Cat# 320881 RNAscope 3-plex Negative control probe Advanced Cell Diagnostics Cat# 320871 (Continued on next page) e2 Neuron 101, 1–18.e1–e13, March 6, 2019

Techniques: Construct, Western Blot, Over Expression, Injection