Review

soluble nlgn3 (OriGene)

OriGene is a verified supplier

OriGene manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

OriGene soluble nlgn3
Soluble Nlgn3, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+nlgn3/Neuroligin+3+(NLGN3)+(NM_018977)+Human+Recombinant+Protein/bio_rxiv__64898__2026__05__14__725216-302-55-57
Average 94 stars, based on 6 article reviews

soluble nlgn3 - by Bioz Stars, 2026-09

94/100 stars

Images

Clone Assay:

Article Title: Multiple Rare Risk Coding Variants in Postsynaptic Density-Related Genes Associated With Schizophrenia Susceptibility.
Article Snippet: .. Human NLGN3, SHANK2, SHANK3, DLGAP1, and DLGAP3 cDNAs were cloned into expression vectors (tGFP and Myc-DDK tagged) using the PrecisionShuttle vector System (OriGene). .. Vectors containing the mutant were generated using a QuikChange R©Lightning Site-Directed Mutagenesis kit (Agilent Technologies).

Article Title: Multiple Rare Risk Coding Variants in Postsynaptic Density-Related Genes Associated With Schizophrenia Susceptibility
Article Snippet: .. Human NLGN3 , SHANK2 , SHANK3 , DLGAP1 , and DLGAP3 cDNAs were cloned into expression vectors (tGFP and Myc-DDK tagged) using the PrecisionShuttle vector System (OriGene). .. Vectors containing the mutant were generated using a QuikChange ® Lightning Site-Directed Mutagenesis kit (Agilent Technologies).

Expressing:

Image Search Results

a. Schematic of glioma membrane isolation, solubilization, and affinity capture by immobilized NLGN3 ectodomains. NLGN3 affinity column is prepared by immobilizing biotinylated (c- terminal AviTag) NLGN3 ectodomains within a streptavidin resin b. Selected top hits from the NLGN3 affinity chromatography assay. Purified membrane proteins captured through NLGN3 affinity chromatography were analyzed by mass spectrometry. Note that both pediatric (DIPGXIII) and adult (MGG8) brain cancers show CSPG4 as a top hit. c. Cartoon representation of CSPG4 functional domains. The laminin-G (LNS) domains of CSPG4 are analogous to those of Neurexins (NRXNs), the canonical binding partners to NLGN3, followed by the signature chondroitin sugar-binding site in the middle of core repeat modules. d. Western blot of human CSF with anti-CSPG4 N-term and anti-NLGN3 antibodies. Control patient CSFs are all from patients with acute lymphocytic leukemia (ALL). Only CSF samples from diffuse midline glioma-bearing patients show both CSPG4 and NLGN3. e. Quantification of bands in d (n = the number of patients = 4/group, mean ± s.e.m).

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. Schematic of glioma membrane isolation, solubilization, and affinity capture by immobilized NLGN3 ectodomains. NLGN3 affinity column is prepared by immobilizing biotinylated (c- terminal AviTag) NLGN3 ectodomains within a streptavidin resin b. Selected top hits from the NLGN3 affinity chromatography assay. Purified membrane proteins captured through NLGN3 affinity chromatography were analyzed by mass spectrometry. Note that both pediatric (DIPGXIII) and adult (MGG8) brain cancers show CSPG4 as a top hit. c. Cartoon representation of CSPG4 functional domains. The laminin-G (LNS) domains of CSPG4 are analogous to those of Neurexins (NRXNs), the canonical binding partners to NLGN3, followed by the signature chondroitin sugar-binding site in the middle of core repeat modules. d. Western blot of human CSF with anti-CSPG4 N-term and anti-NLGN3 antibodies. Control patient CSFs are all from patients with acute lymphocytic leukemia (ALL). Only CSF samples from diffuse midline glioma-bearing patients show both CSPG4 and NLGN3. e. Quantification of bands in d (n = the number of patients = 4/group, mean ± s.e.m).

Article Snippet: DNA sequence encoding the signal peptide and ectodomain of human NLGN3 (Sino Biological NLGN3=HG11160-UT) and subsequently cloned into a mammalian cell expression vector (pD649) with AviTag and 6xHis Tag at the C-terminus.

Techniques: Membrane, Isolation, Affinity Column, Affinity Chromatography, Purification, Mass Spectrometry, Functional Assay, Binding Assay, Western Blot, Control

a. Workflow depicting treatment of glioma cells or OPCs for 1 hour with recombinant NLGN3 ectodomains (to mimic naturally shed NLGN3) followed by conditioned medium (CM) concentration and western blot to detect shedding of CSPG4. b. Left : CSPG4 Western blot from diffuse intrinsic pontine glioma model SU-DIPG13 conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. CSPG4 exists as a ‘part-time’ proteoglycan and is detected as either a core protein (discrete band at ∼268 kDa) or as a chondroitin sulfate proteoglycan (higher molecular weight smear). Treatment of conditioned medium with chondroitinase ABC removes chondroitin sulfate chains and collapses diffuse smears into discrete bands. A c-terminal specific CSPG4 antibody only detects protein in the cell lysate control lane, confirming that conditioned medium bands are shed ectodomains. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.003). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right : Quantification of CSPG4 shedding as fold change in protein band intensities in the left (top N-terminal blot) relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. c. Left : Western blot of CSPG4 shedding from IDH1 WT glioblastoma (GBM) model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0013). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. d. Left : Western blot of CSPG4 shedding from NF1-mutant optic pathway glioma model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0066). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0004). Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the text and the figures. e. Left : Western blot of CSPG4 shedding from primary mouse oligodendrocyte precursor cell (OPC) conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0011). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0003) Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 3, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the figures. f. Model depicting serial events occurring during NLGN3-induced CSPG4 ectodomain shedding by ADAM10 from glioma cells. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain.

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. Workflow depicting treatment of glioma cells or OPCs for 1 hour with recombinant NLGN3 ectodomains (to mimic naturally shed NLGN3) followed by conditioned medium (CM) concentration and western blot to detect shedding of CSPG4. b. Left : CSPG4 Western blot from diffuse intrinsic pontine glioma model SU-DIPG13 conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. CSPG4 exists as a ‘part-time’ proteoglycan and is detected as either a core protein (discrete band at ∼268 kDa) or as a chondroitin sulfate proteoglycan (higher molecular weight smear). Treatment of conditioned medium with chondroitinase ABC removes chondroitin sulfate chains and collapses diffuse smears into discrete bands. A c-terminal specific CSPG4 antibody only detects protein in the cell lysate control lane, confirming that conditioned medium bands are shed ectodomains. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.003). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right : Quantification of CSPG4 shedding as fold change in protein band intensities in the left (top N-terminal blot) relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. c. Left : Western blot of CSPG4 shedding from IDH1 WT glioblastoma (GBM) model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0013). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. d. Left : Western blot of CSPG4 shedding from NF1-mutant optic pathway glioma model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0066). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0004). Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the text and the figures. e. Left : Western blot of CSPG4 shedding from primary mouse oligodendrocyte precursor cell (OPC) conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0011). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0003) Right : Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 3, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the figures. f. Model depicting serial events occurring during NLGN3-induced CSPG4 ectodomain shedding by ADAM10 from glioma cells. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain.

Techniques: Recombinant, Concentration Assay, Western Blot, Molecular Weight, Control, Mutagenesis

a. Workflow depicting treatment of IDH1 WT GBM (MGG8) cells for 1 hour with the potassium channel blocker 4-Aminopyridine (4-AP) followed by culture medium concentration and western blot to detect shedding of CSPG4. b. Left : Representative western blot of shed CSPG4 ectodomains from experiment depicted in a. 4-AP treatment increases shedding relative to vehicle controls. Note that 4-AP-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p < 0.0001) Right : Quantification of western blots. (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. c. Workflow depicting blue light stimulation of Channelrhodopsin-2 (ChR2)-expressing rat OPCs followed by culture medium concentration and western blot to detect shedding of CSPG4. d. Left: Representative current of ChR2-expressing OPCs. (Light power density = 1.0 mW/mm 2 , wavelength = 470 nm, duration = 1 second). Right: Representative western blot of shed CSPG4 ectodomains from experiment depicted in c (pulse width = 25 ms, frequency = 10 Hz). Membrane depolarization by ChR2 stimulation increases shedding relative to no blue light stimulation controls. e. Workflow depicting treatment of IDH1 WT GBM cells (MGG8) for 1-hour with the actin polymerization inhibitor cytochalasin D followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Left : Western blot of shed CSPG4 ectodomains from experiment depicted in e. Cytochalasin D treatment increases shedding relative to vehicle controls (p = 0.0175). Cytochalasin D-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p < 0.0001) Right : Quantification of western blots (n (independent biological replicates of western blot) = 7, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Cartoon diagram summarizing the treatments of 4-AP, ChR2, and cytochalasin D, that are inducing membrane depolarization. h. Model depicting CSPG4 shedding through NLGN3 interaction that implicates an ion channel that is sensitive to membrane stress to depolarize the membrane and activate ADAM10.

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. Workflow depicting treatment of IDH1 WT GBM (MGG8) cells for 1 hour with the potassium channel blocker 4-Aminopyridine (4-AP) followed by culture medium concentration and western blot to detect shedding of CSPG4. b. Left : Representative western blot of shed CSPG4 ectodomains from experiment depicted in a. 4-AP treatment increases shedding relative to vehicle controls. Note that 4-AP-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p < 0.0001) Right : Quantification of western blots. (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. c. Workflow depicting blue light stimulation of Channelrhodopsin-2 (ChR2)-expressing rat OPCs followed by culture medium concentration and western blot to detect shedding of CSPG4. d. Left: Representative current of ChR2-expressing OPCs. (Light power density = 1.0 mW/mm 2 , wavelength = 470 nm, duration = 1 second). Right: Representative western blot of shed CSPG4 ectodomains from experiment depicted in c (pulse width = 25 ms, frequency = 10 Hz). Membrane depolarization by ChR2 stimulation increases shedding relative to no blue light stimulation controls. e. Workflow depicting treatment of IDH1 WT GBM cells (MGG8) for 1-hour with the actin polymerization inhibitor cytochalasin D followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Left : Western blot of shed CSPG4 ectodomains from experiment depicted in e. Cytochalasin D treatment increases shedding relative to vehicle controls (p = 0.0175). Cytochalasin D-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p < 0.0001) Right : Quantification of western blots (n (independent biological replicates of western blot) = 7, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Cartoon diagram summarizing the treatments of 4-AP, ChR2, and cytochalasin D, that are inducing membrane depolarization. h. Model depicting CSPG4 shedding through NLGN3 interaction that implicates an ion channel that is sensitive to membrane stress to depolarize the membrane and activate ADAM10.

Techniques: Concentration Assay, Western Blot, Expressing, Membrane

$a. Representative images of glioma cells colored based on the lifetime of fluorescent tension sensor FlippeR-TR. The fluorescence probe works by specifically targeting the plasma membrane, and the longer lifetime corresponds to increased membrane tension. b. Quantification of the lifetimes measured from the experiment shown in a. (n (number of independent biological samples used for imaging) = 6 – 10, mean ± s.e.m., two-tailed t-test, **** P < 0.0001, n.s. = not significant) c. Scatterplots depicting tumor architecture of scRNA-seq produced from molecularly distinct patient glioma samples. From left to right are Histone H3K27M mutated diffuse midline glioma (DMG) and IDH1 wild-type glioblastoma (GBM). PIEZO1 expression displayed in red across two classes of glioma. Note that PIEZO1 is detected within the OPC-like fraction of these distinct glioma types. d. left: Confocal image of immunostaining in SU-DIPGXIII-FL xenografted mouse brain slice. (PIEZO1 = red, GFP (marker for glioma cells) = green, scale bar = 10 µm) right: 3-dimensional reconstructions of glioma cell surface showing PIEZO1 localization. Patient-derived DIPG cells (SU-DIPGXIII-FL; nestin, blue) co-localize with PIEZO1 puncta (red). Scale bar, 3 µm. e. Workflow depicting treatment of glioma cells with recombinant NLGN3 ectodomains in the presence or absence of modulators of mechanosensitive ion channels (MSC) followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Western blot of shed CSPG4 ectodomains from experiment depicted in c. Treatment with Yoda1 (PIEZO1 agonist), increases shedding relative to vehicle controls (p = 0.0464). In contrast, treating cells with MSC blockers gadolinium (Gd 3+ ) or the spider toxin GsMTx4 prevent NLGN3-induced shedding of CSPG4 (p = 0.0008 and p < 0.0001, respectively) Right : Quantification of western blots (n (independent biological replicates of western blot) = 11, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Model depicting CSPG4 shedding via ADAM10 activation that is downstream of NLGN3 interaction and ensuing membrane depolarization through PIEZO1 channel activation. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.$

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. Representative images of glioma cells colored based on the lifetime of fluorescent tension sensor FlippeR-TR. The fluorescence probe works by specifically targeting the plasma membrane, and the longer lifetime corresponds to increased membrane tension. b. Quantification of the lifetimes measured from the experiment shown in a. (n (number of independent biological samples used for imaging) = 6 – 10, mean ± s.e.m., two-tailed t-test, **** P < 0.0001, n.s. = not significant) c. Scatterplots depicting tumor architecture of scRNA-seq produced from molecularly distinct patient glioma samples. From left to right are Histone H3K27M mutated diffuse midline glioma (DMG) and IDH1 wild-type glioblastoma (GBM). PIEZO1 expression displayed in red across two classes of glioma. Note that PIEZO1 is detected within the OPC-like fraction of these distinct glioma types. d. left: Confocal image of immunostaining in SU-DIPGXIII-FL xenografted mouse brain slice. (PIEZO1 = red, GFP (marker for glioma cells) = green, scale bar = 10 µm) right: 3-dimensional reconstructions of glioma cell surface showing PIEZO1 localization. Patient-derived DIPG cells (SU-DIPGXIII-FL; nestin, blue) co-localize with PIEZO1 puncta (red). Scale bar, 3 µm. e. Workflow depicting treatment of glioma cells with recombinant NLGN3 ectodomains in the presence or absence of modulators of mechanosensitive ion channels (MSC) followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Western blot of shed CSPG4 ectodomains from experiment depicted in c. Treatment with Yoda1 (PIEZO1 agonist), increases shedding relative to vehicle controls (p = 0.0464). In contrast, treating cells with MSC blockers gadolinium (Gd 3+ ) or the spider toxin GsMTx4 prevent NLGN3-induced shedding of CSPG4 (p = 0.0008 and p < 0.0001, respectively) Right : Quantification of western blots (n (independent biological replicates of western blot) = 11, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Model depicting CSPG4 shedding via ADAM10 activation that is downstream of NLGN3 interaction and ensuing membrane depolarization through PIEZO1 channel activation. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Techniques: Fluorescence, Clinical Proteomics, Membrane, Imaging, Two Tailed Test, Produced, Expressing, Immunostaining, Slice Preparation, Marker, Derivative Assay, Recombinant, Concentration Assay, Western Blot, Activation Assay

a. (Left) Schematics of cell-attached patch clamp recording of mechanosensitive currents from glioma cells. (Right) The representative waveforms of mechanosensitive currents measured with 10 mmHg and 30 mmHg pressure inputs. b. Representative traces of mechanosensitive currents (pressure input = 40 mmHg, 1 second) with vehicle (black), PIEZO1 agonist Yoda1 (cyan), NLGN3 (red) and PIEZO1 blocker GsMTx4 (brown). c. Summary of mechanosensitive currents shown in b. (n (number of independent biological samples) = 6 – 8, mean ± s.e.m., Kruskal-Wallis test, ** P < 0.01, *** P < 0.001, n.s. = not significant) d. Schematics of xenografting of SU-DIPGVI WT/PIEZO1 KO into immunocompromised mice followed by assessment of proliferation with Ki67+/HNA staining. e. Representative confocal images of WT and PIEZO1 KO brain slices explained in d. (white = Ki67, red = HNA, white arrows denote the overlapping cells with both Ki67 and HNA stained). f. Summary of tumor proliferation (g, %Ki67+) in WT and PIEZO1 KO samples. (n (number of independent biological samples) = 5, mean ± s.e.m., two-tailed t-test, * P < 0.05). g. Volcano plot of differentially expressed genes comparing WT and PIEZO1 KO MGG8 cells. Genes enriched in PIEZO1 KO relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in PIEZO1 KO cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p- value). h. Gene ontology analysis of genes that were significantly depleted in PIEZO1 KO cells relative to WT. Enriched programs all center around sterol biosynthesis and metabolism. Of note, FABP7 is amongst the most strongly down-regulated genes in PIEZO1 KO cells and is reported to function in the maintenance of both glioma stem cells and healthy OPCs . i. Cartoon model depicting mechanism of NLGN3-induced CSPG4 shedding and functional consequences. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. (Left) Schematics of cell-attached patch clamp recording of mechanosensitive currents from glioma cells. (Right) The representative waveforms of mechanosensitive currents measured with 10 mmHg and 30 mmHg pressure inputs. b. Representative traces of mechanosensitive currents (pressure input = 40 mmHg, 1 second) with vehicle (black), PIEZO1 agonist Yoda1 (cyan), NLGN3 (red) and PIEZO1 blocker GsMTx4 (brown). c. Summary of mechanosensitive currents shown in b. (n (number of independent biological samples) = 6 – 8, mean ± s.e.m., Kruskal-Wallis test, ** P < 0.01, *** P < 0.001, n.s. = not significant) d. Schematics of xenografting of SU-DIPGVI WT/PIEZO1 KO into immunocompromised mice followed by assessment of proliferation with Ki67+/HNA staining. e. Representative confocal images of WT and PIEZO1 KO brain slices explained in d. (white = Ki67, red = HNA, white arrows denote the overlapping cells with both Ki67 and HNA stained). f. Summary of tumor proliferation (g, %Ki67+) in WT and PIEZO1 KO samples. (n (number of independent biological samples) = 5, mean ± s.e.m., two-tailed t-test, * P < 0.05). g. Volcano plot of differentially expressed genes comparing WT and PIEZO1 KO MGG8 cells. Genes enriched in PIEZO1 KO relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in PIEZO1 KO cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p- value). h. Gene ontology analysis of genes that were significantly depleted in PIEZO1 KO cells relative to WT. Enriched programs all center around sterol biosynthesis and metabolism. Of note, FABP7 is amongst the most strongly down-regulated genes in PIEZO1 KO cells and is reported to function in the maintenance of both glioma stem cells and healthy OPCs . i. Cartoon model depicting mechanism of NLGN3-induced CSPG4 shedding and functional consequences. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Techniques: Patch Clamp, Staining, Two Tailed Test, Functional Assay

a. Workflow depicting EdU incorporation and detection in rat OPCs following treatment with NLGN3. b. Representative images of EdU incorporation in primary rat OPCs after 4-hour treatments. c. Quantification of cell proliferation depicted in a. (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. **** P < 0.0001) d. Schematic depicting differentiation and morphological changes of OPCs upon mitogen withdrawal in vitro . e. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3. f. Quantification of cells at each morphological stage following 48-hours of treatment. ( n (number of independent trials) = 4, two-way ANOVA with multiple comparisons and Bonferroni correction, not significant) Note that PDGFAA treated and NLGN3-treated cells are not significantly different regarding progression to stage 3 morphology as assessed by two-way ANOVA with multiple comparisons and Bonferroni correction. g. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3 and ADAM10 inhibition. h. Quantification of OPC (CSPG4 positive) to OL (MBP positive) ratio in cells depicted in g (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. p-values are written on the figures). i. PCA plot of bulk RNA-seq produced from rat OPCs that were cultured for 48 hours under PDGFAA withdrawal and supplementation with NLGN3 and/or inhibitors of CSPG4 shedding. Vehicle and NLGN3-treated cells are distinct in PC space from the inhibitor-treated samples. j. Volcano plot of differentially expressed genes comparing NLGN3-treated rat OPCs against PDGFAA withdrawal alone. Genes enriched in NLGN3-treated relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in NLGN3-treated cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p-value). k. Heatmap of NLGN3-induced genes from Venkatesh et al 2017. Primary rat OPCs were cultured for 48 hours without PDGFAA to initiate differentiation and NLGN3 was supplemented in half the cultures. Duplicate vehicle and NLGN3-treated samples were profiled by bulk RNA-seq. TPM values for each condition were averaged and then the log2 fold change was calculated between NLGN3-treated and vehicle control samples. These values were plotted such that zero appears as white on a symmetrical axis that ranges from -4 in blue and 4 in red. l. Model depicting CSPG4 shedding via PIEZO1 opening and ADAM10 activation. These events impede OPC differentiation in vitro upon growth factor withdrawal. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Journal: bioRxiv

Article Title: Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

doi: 10.1101/2025.07.12.664340

Figure Lengend Snippet: a. Workflow depicting EdU incorporation and detection in rat OPCs following treatment with NLGN3. b. Representative images of EdU incorporation in primary rat OPCs after 4-hour treatments. c. Quantification of cell proliferation depicted in a. (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. **** P < 0.0001) d. Schematic depicting differentiation and morphological changes of OPCs upon mitogen withdrawal in vitro . e. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3. f. Quantification of cells at each morphological stage following 48-hours of treatment. ( n (number of independent trials) = 4, two-way ANOVA with multiple comparisons and Bonferroni correction, not significant) Note that PDGFAA treated and NLGN3-treated cells are not significantly different regarding progression to stage 3 morphology as assessed by two-way ANOVA with multiple comparisons and Bonferroni correction. g. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3 and ADAM10 inhibition. h. Quantification of OPC (CSPG4 positive) to OL (MBP positive) ratio in cells depicted in g (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. p-values are written on the figures). i. PCA plot of bulk RNA-seq produced from rat OPCs that were cultured for 48 hours under PDGFAA withdrawal and supplementation with NLGN3 and/or inhibitors of CSPG4 shedding. Vehicle and NLGN3-treated cells are distinct in PC space from the inhibitor-treated samples. j. Volcano plot of differentially expressed genes comparing NLGN3-treated rat OPCs against PDGFAA withdrawal alone. Genes enriched in NLGN3-treated relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in NLGN3-treated cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p-value). k. Heatmap of NLGN3-induced genes from Venkatesh et al 2017. Primary rat OPCs were cultured for 48 hours without PDGFAA to initiate differentiation and NLGN3 was supplemented in half the cultures. Duplicate vehicle and NLGN3-treated samples were profiled by bulk RNA-seq. TPM values for each condition were averaged and then the log2 fold change was calculated between NLGN3-treated and vehicle control samples. These values were plotted such that zero appears as white on a symmetrical axis that ranges from -4 in blue and 4 in red. l. Model depicting CSPG4 shedding via PIEZO1 opening and ADAM10 activation. These events impede OPC differentiation in vitro upon growth factor withdrawal. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Techniques: In Vitro, Inhibition, RNA Sequencing, Produced, Cell Culture, Control, Activation Assay

Fig. 3 | BDNF regulates trafficking of AMPAR to the glioma postsynaptic membrane. a, Schematic depicting AMPAR trafficking downstream of BDNF– TrkB–CAMKII signalling46. b, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c, Quantification of cell surface GluA4 in b (n = 3 independent biological replicates). d, Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e, Quantification of cell surface GluA3 in d (n = 3 independent biological replicates). f, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g, Quantification of cell surface GluA4 data in f (n = 3 independent biological replicates). h, Schematic showing GluA2–SEP experiments. i,j, Validation of pHluorin approach. i, Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity.

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: Fig. 3 | BDNF regulates trafficking of AMPAR to the glioma postsynaptic membrane. a, Schematic depicting AMPAR trafficking downstream of BDNF– TrkB–CAMKII signalling46. b, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c, Quantification of cell surface GluA4 in b (n = 3 independent biological replicates). d, Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e, Quantification of cell surface GluA3 in d (n = 3 independent biological replicates). f, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g, Quantification of cell surface GluA4 data in f (n = 3 independent biological replicates). h, Schematic showing GluA2–SEP experiments. i,j, Validation of pHluorin approach. i, Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture

Article Snippet: Recombinant BDNF human protein (Peprotech, 450-02), or NLGN3 (OriGene Technologies, TP307955), was added to ACSF at 100 ng ml−1 and perfused for 30 min to test changes in response to glutamate puff or evoked stimulation.

Techniques: Membrane, Western Blot, Biomarker Discovery, Expressing, Co-Culture Assay

Fig. 3. Two novel NLGN3 nonsense variants found in patients with GnRH deficiency (GD) and autism spectrum disorder (ASD). (A) Family pedigrees of the two probands (Case 1 and Case 2) identified in this study and presenting GD and ASD features. Sequence chromatograms of nucleotides 70367955-70367975 and nucleotides 70367752-70367772 of the NLGN3 coding sequence in Case 1, Case 2 and their unaffected mothers (the positions of the C>T and G>A are highlighted by the red boxes). (B) Schematic representation of NLGN3 protein (encoded by the NM_181303.1 transcript): signal peptide (light blue), extracellular (green), transmembrane (yellow) and intracellular (orange) domains. All missense variants (gray dots) reported in gnomAD database (v2.1.1) are plotted according to their amino acid (aa) position and Combined Annotation-Dependent Depletion (CADD) score. Pathogenic variants related to ASD are indicated in black: R471C (Jamain et al., 2003), P534S (Quartier et al., 2019) and R617W (Redin et al., 2014). Identified variants (R55* and W122*) are indicated in red and have a higher CADD score compared to others. Multi-species alignment of partial protein sequences of vertebrate NLGN3 ortholog proteins shows that the R55 and W122 residues are evolutionarily conserved in humans and other vertebrate species with a high conservation degree, calculated by GERP++.

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 3. Two novel NLGN3 nonsense variants found in patients with GnRH deficiency (GD) and autism spectrum disorder (ASD). (A) Family pedigrees of the two probands (Case 1 and Case 2) identified in this study and presenting GD and ASD features. Sequence chromatograms of nucleotides 70367955-70367975 and nucleotides 70367752-70367772 of the NLGN3 coding sequence in Case 1, Case 2 and their unaffected mothers (the positions of the C>T and G>A are highlighted by the red boxes). (B) Schematic representation of NLGN3 protein (encoded by the NM_181303.1 transcript): signal peptide (light blue), extracellular (green), transmembrane (yellow) and intracellular (orange) domains. All missense variants (gray dots) reported in gnomAD database (v2.1.1) are plotted according to their amino acid (aa) position and Combined Annotation-Dependent Depletion (CADD) score. Pathogenic variants related to ASD are indicated in black: R471C (Jamain et al., 2003), P534S (Quartier et al., 2019) and R617W (Redin et al., 2014). Identified variants (R55* and W122*) are indicated in red and have a higher CADD score compared to others. Multi-species alignment of partial protein sequences of vertebrate NLGN3 ortholog proteins shows that the R55 and W122 residues are evolutionarily conserved in humans and other vertebrate species with a high conservation degree, calculated by GERP++.

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (fw, 5′-GGCAGTGGTACTCAGGCACCCCTTAGC-3′; rev, 5′-GCTAAGGGGTGCCTGAGTACCACTGCC-3′) and W122* (fw, 5′-GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3′; rev, 5′-TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3′).

Techniques: Sequencing

Fig. 4. NLGN3 is developmentally regulated in GnRH neurons. (A) Quantitative PCR analysis performed on GN11 and GT1-7 cells revealed higher Nlgn3 expression levels in GT1-7 cells (logFC=8.12, P<0.01). This result is in line with microarray experiments (logFC=4.15; P<0.00001). Data are presented as mean±s.d. of three biological replicates. Unpaired two-tailed Student’s t-test (**P<0.01). (B) Immunoperoxidase staining for NLGN3 on GN11 and GT1-7 revealed different levels of endogenous NLGN3 protein in these cells. Scale bar: 25 μm. (C,D) Coronal sections of E14.5 mouse heads were immunolabeled for NLGN3 together with GnRH (C) or PLXND1 (D) to detect GnRH neurons. Sections are shown at the level of the VNO (nose; C) or MPOA (forebrain; D). White dashed line boxes indicate areas shown at higher magnification on the right of the corresponding panel, with single channels also shown adjacent to the panel. Open arrowheads indicate examples of GnRH-positive cells that lack NLGN3; filled arrowheads indicate examples of GnRH-positive cells with NLGN3. Arrows indicate examples of NLGN3- positive cells in the nasal parenchyma. Sections were counterstained with DAPI. MPOA, medial preoptic area; OB, olfactory bulb; OE, olfactory epithelium; VNO, vomeronasal organ. Scale bars: 250 μm (right panels), 150 μm (middle panels) or 50 μm (left panels).

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 4. NLGN3 is developmentally regulated in GnRH neurons. (A) Quantitative PCR analysis performed on GN11 and GT1-7 cells revealed higher Nlgn3 expression levels in GT1-7 cells (logFC=8.12, P<0.01). This result is in line with microarray experiments (logFC=4.15; P<0.00001). Data are presented as mean±s.d. of three biological replicates. Unpaired two-tailed Student’s t-test (**P<0.01). (B) Immunoperoxidase staining for NLGN3 on GN11 and GT1-7 revealed different levels of endogenous NLGN3 protein in these cells. Scale bar: 25 μm. (C,D) Coronal sections of E14.5 mouse heads were immunolabeled for NLGN3 together with GnRH (C) or PLXND1 (D) to detect GnRH neurons. Sections are shown at the level of the VNO (nose; C) or MPOA (forebrain; D). White dashed line boxes indicate areas shown at higher magnification on the right of the corresponding panel, with single channels also shown adjacent to the panel. Open arrowheads indicate examples of GnRH-positive cells that lack NLGN3; filled arrowheads indicate examples of GnRH-positive cells with NLGN3. Arrows indicate examples of NLGN3- positive cells in the nasal parenchyma. Sections were counterstained with DAPI. MPOA, medial preoptic area; OB, olfactory bulb; OE, olfactory epithelium; VNO, vomeronasal organ. Scale bars: 250 μm (right panels), 150 μm (middle panels) or 50 μm (left panels).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Microarray, Two Tailed Test, Immunoperoxidase Staining, Immunolabeling

Fig. 5. Mutant NLGN3 proteins induce endoplasmic reticulum retention and impair neuritogenesis in immortalized GnRH neurons. (A) Immunoblot analysis with anti-HA or anti-GAPDH antibodies on whole-cell lysates and conditioned media from COS7 cells overexpressing control vector (GFP), HA- tagged wild-type (WT) or mutant NLGN3. GAPDH is shown as a loading control for cell lysates. (B) Confocal images of GN11 cells transfected cells with mEmerald-ER-3 (green) and human WT or mutated HA-tagged NLGN3 vectors and stained for HA (red) after 24 h. HA-NLGN3 single-channel images are shown below each panel. (C) Confocal images of GN11 transfected with human WT or mutated NLGN3 HA-tagged encoding vector and stained for HA (green) and F-actin (red). Arrowheads point at neurites in NLGN3 WT-expressing GN11 cells. (D) Quantification of cell perimeter (P), cell area (A), aspect ratio (AR) and complexity index (CI) in GN11 cells transfected with indicated plasmids. Column graph quantifications show a significant increase in the number of neurites extending from NLGN3 WT-expressing cells compared to NLGN3 mutants and GFP-transfected control cells. Data are presented as mean±s.d. of three biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test (***P<0.001; ****P<0.0001). Nuclei were counterstained with DAPI. Scale bars: 25 μm.

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 5. Mutant NLGN3 proteins induce endoplasmic reticulum retention and impair neuritogenesis in immortalized GnRH neurons. (A) Immunoblot analysis with anti-HA or anti-GAPDH antibodies on whole-cell lysates and conditioned media from COS7 cells overexpressing control vector (GFP), HA- tagged wild-type (WT) or mutant NLGN3. GAPDH is shown as a loading control for cell lysates. (B) Confocal images of GN11 cells transfected cells with mEmerald-ER-3 (green) and human WT or mutated HA-tagged NLGN3 vectors and stained for HA (red) after 24 h. HA-NLGN3 single-channel images are shown below each panel. (C) Confocal images of GN11 transfected with human WT or mutated NLGN3 HA-tagged encoding vector and stained for HA (green) and F-actin (red). Arrowheads point at neurites in NLGN3 WT-expressing GN11 cells. (D) Quantification of cell perimeter (P), cell area (A), aspect ratio (AR) and complexity index (CI) in GN11 cells transfected with indicated plasmids. Column graph quantifications show a significant increase in the number of neurites extending from NLGN3 WT-expressing cells compared to NLGN3 mutants and GFP-transfected control cells. Data are presented as mean±s.d. of three biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test (***P<0.001; ****P<0.0001). Nuclei were counterstained with DAPI. Scale bars: 25 μm.

Techniques: Mutagenesis, Western Blot, Control, Plasmid Preparation, Transfection, Staining, Expressing, Comparison

A - Family pedigrees of the two probands (case 1 and case 2) identified in this study and presenting GD and ASD features. Sequence chromatograms of nucleotides 70367955-70367975 of the NLGN3 coding sequence in proband 1 and his unaffected parent (the position of the G>A is highlighted by the red box). B - Schematic representation of NLGN3 protein (encoded by the NM_181303.1 transcript): signal peptide (sp, light blue), extracellular (green), transmembrane (tm, yellow) and intracellular (red) domains. All missense variants (grey dots) reported in gnomAD database (v2.1.1) are plotted according to their aminoacidic position and CADD score. Pathogenic variants related to ASD are indicated in black: R471C , P534S and R617W . Identified variants (R55* and W122*) are indicated in red and have a higher CADD score compared to others. Multi-species alignment of partial protein sequences of vertebrate NLGN3 orthologue proteins shows that the R55 and W122 residues are evolutionarily conserved in humans and other vertebrate species with a high conservation degree, calculated by GERP++.

Journal: medRxiv

Article Title: Combined omic analyses reveal novel loss-of-function NLGN3 variants in GnRH deficiency and autism

doi: 10.1101/2022.05.24.22275221

Figure Lengend Snippet: A - Family pedigrees of the two probands (case 1 and case 2) identified in this study and presenting GD and ASD features. Sequence chromatograms of nucleotides 70367955-70367975 of the NLGN3 coding sequence in proband 1 and his unaffected parent (the position of the G>A is highlighted by the red box). B - Schematic representation of NLGN3 protein (encoded by the NM_181303.1 transcript): signal peptide (sp, light blue), extracellular (green), transmembrane (tm, yellow) and intracellular (red) domains. All missense variants (grey dots) reported in gnomAD database (v2.1.1) are plotted according to their aminoacidic position and CADD score. Pathogenic variants related to ASD are indicated in black: R471C , P534S and R617W . Identified variants (R55* and W122*) are indicated in red and have a higher CADD score compared to others. Multi-species alignment of partial protein sequences of vertebrate NLGN3 orthologue proteins shows that the R55 and W122 residues are evolutionarily conserved in humans and other vertebrate species with a high conservation degree, calculated by GERP++.

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (FW 5’- GGCAGTGGTACTCAGGCACCCCTTAGC-3’, RV 5’- GCTAAGGGGTGCCTGAGTACCACTGCC-3’) and W122* (FW 5’- GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3’, RV 5’- TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3’) respectively.

Techniques: Sequencing

Journal: medRxiv

Article Title: Combined omic analyses reveal novel loss-of-function NLGN3 variants in GnRH deficiency and autism

doi: 10.1101/2022.05.24.22275221

Figure Lengend Snippet:

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (FW 5’- GGCAGTGGTACTCAGGCACCCCTTAGC-3’, RV 5’- GCTAAGGGGTGCCTGAGTACCACTGCC-3’) and W122* (FW 5’- GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3’, RV 5’- TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3’) respectively.

Techniques:

A - qPCR analysis performed on GN11 and GT1-7 cells revealed higher Nlgn3 expression levels in GT1-7 cells (log FC = 8.12, P < 0.01). This result is in line with microarray experiments (log FC = 4.15; P < 0.00001). Data are presented as mean ± SD of 3 biological replicates. P values indicate Student’s t test (** P < 0.01). B - Immunoperoxidase staining for NLGN3 on GN11 and GT1- 7 revealed different levels of endogenous NLGN3 protein in these cells. Scale bar: 25 μm. C,D - Coronal sections of E14.5 mouse heads were immunolabelled for NLGN3 together with GnRH or PLXND1 to detect GnRH neurons. Sections are shown at the level of the VNO (nose, C) or MPOA (forebrain, D). White boxes indicate areas shown at higher magnification on the right of the corresponding panel, with single channels shown also adjacent to the panel. Open arrowheads indicate examples of GnRH-positive cells that lack NLGN3. Arrows indicate examples of NLGN3-positive cells in the nasal parenchyma (C). Arrowheads indicate examples of GnRH-positive cells with NLGN3 (D). Sections were counterstained with DAPI. OE, olfactory epithelium; OB, olfactory bulb; VNO, vomeronasal organ; MPOA, medial preoptic area. Scale bars: 250 (right panels), 150 (middle panels) or 50 μm (left panels).

Journal: medRxiv

Article Title: Combined omic analyses reveal novel loss-of-function NLGN3 variants in GnRH deficiency and autism

doi: 10.1101/2022.05.24.22275221

Figure Lengend Snippet: A - qPCR analysis performed on GN11 and GT1-7 cells revealed higher Nlgn3 expression levels in GT1-7 cells (log FC = 8.12, P < 0.01). This result is in line with microarray experiments (log FC = 4.15; P < 0.00001). Data are presented as mean ± SD of 3 biological replicates. P values indicate Student’s t test (** P < 0.01). B - Immunoperoxidase staining for NLGN3 on GN11 and GT1- 7 revealed different levels of endogenous NLGN3 protein in these cells. Scale bar: 25 μm. C,D - Coronal sections of E14.5 mouse heads were immunolabelled for NLGN3 together with GnRH or PLXND1 to detect GnRH neurons. Sections are shown at the level of the VNO (nose, C) or MPOA (forebrain, D). White boxes indicate areas shown at higher magnification on the right of the corresponding panel, with single channels shown also adjacent to the panel. Open arrowheads indicate examples of GnRH-positive cells that lack NLGN3. Arrows indicate examples of NLGN3-positive cells in the nasal parenchyma (C). Arrowheads indicate examples of GnRH-positive cells with NLGN3 (D). Sections were counterstained with DAPI. OE, olfactory epithelium; OB, olfactory bulb; VNO, vomeronasal organ; MPOA, medial preoptic area. Scale bars: 250 (right panels), 150 (middle panels) or 50 μm (left panels).

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (FW 5’- GGCAGTGGTACTCAGGCACCCCTTAGC-3’, RV 5’- GCTAAGGGGTGCCTGAGTACCACTGCC-3’) and W122* (FW 5’- GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3’, RV 5’- TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3’) respectively.

Techniques: Expressing, Microarray, Immunoperoxidase Staining

A - Immunoblot analysis with anti-HA or anti-GAPDH antibodies on whole-cell lysates and conditioned media from COS7 cells overexpressing control vector (GFP), HA-tagged WT or mutant NLGN3. GAPDH is shown as a loading control for cell lysates. B - Confocal images of GN11 cells transfected cells with mEmerald-ER-3 (green) and human WT or mutated HA-tagged NLGN3 vectors and stained for HA (red) after 24 h (right panels). HA-NLGN3 single channel images are shown below each panel. C - Confocal images of GN11 transfected with human WT or mutated NLGN3 HA-tagged encoding vector and stained for HA (green) and F- actin (red). Arrowheads point at neurites in NLGN3 WT-expressing GN11 cells. D - Quantification of cell perimeter (P), cell area (A), aspect ratio (AR) and complexity index (CI) in GN11 cells transfected with indicated plasmids. Column graph quantification showing a significant increase in the number of neurites extending from NLGN3 WT-expressing cells compared to NLGN3 mutants and GFP-transfected control cells. Data are presented as mean ± SD of 3 biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test (***P < 0.001; ****P<0.0001). Nuclei were counterstained with DAPI. Scale bar: 25 μm.

Journal: medRxiv

Article Title: Combined omic analyses reveal novel loss-of-function NLGN3 variants in GnRH deficiency and autism

doi: 10.1101/2022.05.24.22275221

Figure Lengend Snippet: A - Immunoblot analysis with anti-HA or anti-GAPDH antibodies on whole-cell lysates and conditioned media from COS7 cells overexpressing control vector (GFP), HA-tagged WT or mutant NLGN3. GAPDH is shown as a loading control for cell lysates. B - Confocal images of GN11 cells transfected cells with mEmerald-ER-3 (green) and human WT or mutated HA-tagged NLGN3 vectors and stained for HA (red) after 24 h (right panels). HA-NLGN3 single channel images are shown below each panel. C - Confocal images of GN11 transfected with human WT or mutated NLGN3 HA-tagged encoding vector and stained for HA (green) and F- actin (red). Arrowheads point at neurites in NLGN3 WT-expressing GN11 cells. D - Quantification of cell perimeter (P), cell area (A), aspect ratio (AR) and complexity index (CI) in GN11 cells transfected with indicated plasmids. Column graph quantification showing a significant increase in the number of neurites extending from NLGN3 WT-expressing cells compared to NLGN3 mutants and GFP-transfected control cells. Data are presented as mean ± SD of 3 biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test (***P < 0.001; ****P<0.0001). Nuclei were counterstained with DAPI. Scale bar: 25 μm.

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (FW 5’- GGCAGTGGTACTCAGGCACCCCTTAGC-3’, RV 5’- GCTAAGGGGTGCCTGAGTACCACTGCC-3’) and W122* (FW 5’- GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3’, RV 5’- TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3’) respectively.

Techniques: Western Blot, Plasmid Preparation, Mutagenesis, Transfection, Staining, Expressing

Review

soluble nlgn3 (OriGene)

Structured Review

Images

Related Articles

Clone Assay:

Expressing:

Similar Products

Image Search Results