Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: Effects of downregulated JMJD3 on EC survival and migration. A , ECs infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl) for 2 days at 90% confluence in a 12-well plate. Western blot detected the expressions of JMJD3, UTX, PCNA and methylation of H3K27. Pictures are representative of three independent experiments. B , quantification of Western blot analyses of the expressions of JMJD3 and PCNA. Statistical significance was analyzed by one-way ANOVA. C , ECs infected with lentivirus-mediated overexpression of JMJD3 (JMJD3OE) for 2 days at 90% confluence in a 12-well plate. ECs infected with lentivirus packaging with empty vector were used as negative control (Ctl). Western blot was used to detect the expressions of PCNA and methylation of H3K27. Photos are representative of three independent experiments. D , quantification of Western blot analyses in B . Statistical significance was analyzed by one-way ANOVA. E , ECs were cultured in 96-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). EC proliferation was determined by MTS during different time points. Data are presented as mean ± SD from three replicates. Statistical significance between groups was analyzed by two-way ANOVA. F and G , ECs were cultured in 12-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). Wound-healing assay was used to detect the EC migration ( F ). The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch toward the center of the scratch at 2 days or 4 days when compared with 0 days ( G ). EC, endothelial cell; JMJD3, Jumonji domain–containing protein-3; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; sgRNA, single-guide RNA; UTX, ubiquitously transcribed tetratricopeptide repeat, X chromosome.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Migration, Infection, Western Blot, Methylation, Over Expression, Plasmid Preparation, Negative Control, Cell Culture, Wound Healing Assay

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: JMJD3 mediates TGFβ1-induced EndMT. A , Western blot analysis of ECs overexpressing JMJD3 by lentivirus (JMJD3OE) for 2 days following treatment with 2 ng/ml TGFβ1 or solvent for another 5 days. ECs infected with lentivirus packaging with empty vector were used as negative control (Ctl). Photos are representative of three independent experiments. B , quantification of the expression of JMJD3 and H3K27me3 is shown as means ± SD. Statistical significance between two groups was analyzed by one-way ANOVA. C , quantification of the expression of VE-cadherin and α-SMA is shown as means ± SD. Statistical significance between groups was analyzed by two-way ANOVA. D and E , ECs were treated with 2 ng/ml TGFβ1 or solvent (Ctl) for 24 h. ChIP assays with rabbit anti-H3K27me3 or anti-H3K4me3 antibody were performed. Rabbit IgG was used as control. Real-time PCR amplified VE-cadherin promoter with specific primers, respectively. Quantitative PCR data were normalized to IgG negative control and displayed as fold enrichment and expressed as means ± SD. Statistical significance between groups was analyzed by one-way ANOVA. α-SMA, α-smooth muscle actin; ChIP, chromatin immunoprecipitation; EC, endothelial cell; EndMT, endothelial–mesenchymal transition; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; TGFβ1, transforming growth factor beta 1; VE-cadherin, vascular endothelial-cadherin.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Western Blot, Solvent, Infection, Plasmid Preparation, Negative Control, Expressing, Control, Real-time Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: Effects of JMJD3 knockdown in ECs on proliferation of VSMC. A , VSMC cells were plated on the bottom chamber of 96-well transwell cell culture system. ECs were cultured onto the top chamber of the transwell inserts and transduced with lentivirus to knockdown JMJD3 by sgRNA (sgJD) or overexpress JMJD3 (JDOE). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. MTS assay was performed to detect cell proliferation after coculture for 72 h. Data are presented as mean ± SD for three replicates. B – D , VSMCs were cultured in 96-well plate or 12-well plate. SNP (ranged from 1 to 1000 μM) was added to treat VSMC for 48 h. VSMC proliferation was detected by MTS assay. Data are presented as mean ± SD from three replicates ( B ). PCNA expression was evaluated by Western blot ( C ) and density was quantified ( D ). E , ECs were cultured in 12-well plate and treated with lentivirus-mediated sgRNA targeting JMJD3 (sgJD) or overexpressed JMJD3 (JDOE) by lentivirus combined with or without L-NAME (300 μM). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. Expression levels of JMJD3 and eNOS were detected by Western blot. Photos are representative of three independent experiments. F , quantification of Western blot analysis for eNOS is shown as mean ± SD. G , quantitative ChIP analysis of H3K27me3 at the eNOS promoter. ECs were transduced with lentivirus sgRNA targeting JMJD3 (sgJD) or control lentivirus nonspecific sgRNA (Ctl). ChIP assay was performed with rabbit anti-H3K27me3 antibodies. Rabbit IgG was used as control. Real-time PCR amplified eNOS promoter with specific primers. Quantitative PCR data were normalized to IgG negative control and displayed as fold enrichment and expressed as means ± SD. Statistical significance between groups was analyzed by two-way ANOVA. H , VSMCs were cocultured with ECs. VSMCs were plated on the bottom chamber of 12-well transwell cell culture system. ECs were cultured on the top chamber of transwell inserts and transduced with lentivirus to knockdown JMJD3 by sgRNA (sgJD) or overexpress JMJD3 (JDOE) with or without addition of SNP (100 μM) or L-NAME (300 μM). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. After coculture for 48 h, VSMC lysates were used for detecting the expression levels of PCNA by Western blot. VSMC without EC coculture was also used as control (W/O). Photos are representative of three independent experiments. I , quantification of Western blot analysis for PCNA is shown as mean ± SD. All statistical significances between groups were analyzed by one-way ANOVA. ChIP, chromatin immunoprecipitation; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; L-NAME, N (omega)-nitro- l -arginine methyl ester; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; sgRNA, single-guide RNA; SNP, sodium nitroprusside; VSMC, vascular smooth muscle cell.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Knockdown, Cell Culture, Transduction, Control, Plasmid Preparation, MTS Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: JMJD3 is regulated by TGFβ1–Hes1 pathway in ECs. A , real-time PCR analysis of the mRNA level of JMJD3 after treatment of TGFβ1 or solvent (Ctl) for 24 h. Values of JMJD3 expression level were normalized to GAPDH . Data are presented as mean ± SD of three independent experiments. Statistical significance was measured by one-way ANOVA. B , ECs were stimulated with various concentration of TGFβ1 for 48 h. The expression of JMJD3 and methylation of H3K27 was determined by Western blot. C , quantification of Western blot analyses for JMJD3 is shown as mean ± SD. Statistical significance between groups was analyzed by one-way ANOVA. D , scheme of the mouse JMJD3 locus indicating two HES1-binding sites ( vertical arrows ), which were analyzed using JASPAR. Promoter region was used in the luciferase assay. E , Western blot of Hes1 in EC treatment with 2 ng/ml TGFβ1 or solvent for 24 h. Photos are representative of three independent experiments. F , quantification of Western blot analyses for Hes1 is shown as mean ± SD. Statistical significance between groups was analyzed by Mann–Whitney test. G , ECs were transduced with lentivirus overexpressing Hes1 (Hes OE) or empty vector (Ctl). ECs without transduction were also used as control (blank). Western blot was performed after 2 days of culture. Photos are representative of three independent experiments. H , quantification of Western blot analyses for Hes1, JMJD3, and H3K27me3 is shown as mean ± SD. Statistical significance between groups was analyzed by one-way ANOVA. I , ChIP assays with anti-Hes1 or anti-IgG antibody from ECs with 2 ng/ml TGFβ1 or solvent (Ctl) treatment for 24 h. JMJD3 promoter was amplified with specific primers by PCR with 2% input as template for 20 cycles or with immunoprecipitated DNA as template for 35 cycles. Photos are representative of three independent experiments. J , quantification of PCR products for JMJD3 promoter was analyzed by ImageJ and shown as mean ± SD. Statistical significance between groups was analyzed by two-way ANOVA. K , HEK293T cells were cotransfected with JMJD3 promoter and Hes1 recombinant plasmid or control plasmid. Luciferase activity of the JMJD3 promoter was compared in the presence and absence of Hes1. Data are presented as mean ± SD. Spots are presented as values of three replicates. Statistical significance between groups was analyzed by two-way ANOVA. ChIP, chromatin immunoprecipitation; EC, endothelial cell; HEK293T, human embryonic kidney 293T cell line; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; TGFβ1, transforming growth factor beta 1.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Real-time Polymerase Chain Reaction, Solvent, Expressing, Concentration Assay, Methylation, Western Blot, Binding Assay, Luciferase, MANN-WHITNEY, Transduction, Plasmid Preparation, Control, Amplification, Immunoprecipitation, Recombinant, Activity Assay, Chromatin Immunoprecipitation

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: Impact of lineage-enriched transcription factors on expression of markers genes in Pit-1/Triple cells. Pit-1/Triple cells (5) were transfected with expression constructs encoding each noted transcription factor linked by an IRES to a GFP reporter open reading frame (see “Materials and Methods”). Transfected cells were isolated by GFP FACS and their RNA content was assayed for specific marker gene expression by qRT-PCR. (A) Nupr1. (B) Rxrg. (C) Nr4a2. (D) Pou4f1. In all assays, expression of the recombinant transcription factor was confirmed by targeted qRT-PCR (data not shown). Gene expression was normalized to an internal Gapdh control, and the fold change over empty vector controls was calculated using the ΔΔCt method (see “Materials and Methods”). Gapdh expression levels were stable across all assayed conditions, with no significant changes in Gapdh expression in any of the transfected samples. Error bars indicate 1 SD. n = 5 for all experiments. *P < 0.05, by t test.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Transfection, Construct, Isolation, Marker, Gene Expression, Quantitative RT-PCR, Recombinant, Control, Plasmid Preparation

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: NR4A2 acts in conjunction with POU1F1 at the Prl promoter to enhance Prl expression. (A) Schematic of the mPrl gene promoter indicating the relative positions of known POU1F1 binding sites [blue ovals (15)] as well as predicted binding sites for NR4A2 (red ovals) (identified by JASPAR). (B) NR4A2 ChIP. Pit-1/Triple cells were transfected with an expression vector encoding NR4A2, and chromatin isolated from GFP+ cells (as in Fig. 4) was assayed by NR4A2 ChIP. Levels of transcription factor occupancy at specific sites within the Prl promoter were quantified by qRT-PCR of immunoprecipitated samples. Parallel controls included quantitation of occupancy at the Gh promoter and assessment of binding in regions 500 bp upstream and downstream of the Prl promoter. The MyoD promoter served as a negative control in all studies. (C) Schematic of the mPrl gene promoter indicating the relative positioning of known POU1F1 binding sites (blue ovals) as well as predicted binding sites for POU4F1 (green ovals) (identified by JASPAR). (D) POU4F1 ChIP. Pit-1/Triple cells were transfected with an expression vector encoding POU4F1 and chromatin isolated from GFP+ cells [as in (B)] was assayed by ChIP. The Pou4f1 enhancer was assayed in this study as a positive control for the POU4F1 ChIP. (E) Nr4a2 ChIP performed in FACS-sorted mouse lactotropes. (F) Impact of NR4A2 on expression of markers genes in Pit-1/0 cells. Pit-1/0 cells were transfected with expression vectors encoding Pou1f1, Nr4a2, or both linked by IRES to GFP, and GFP+ cells were collected by FACS. qRT-PCR analysis was performed to measure changes in mRNA expression of somatotrope and lactotrope marker genes. n = 3 for all experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Immunoprecipitation, Quantitation Assay, Negative Control, Positive Control, Marker

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: ChIP of POU1F1 in Pit-1/Triple cells expressing Nr4a2 vs Pit-1/Triple cells that do not express Nr4a2. Pit-1/Triple cells, which do not express Nr4a2, were transfected with the same Nr4a2-IRES-GFP plasmid used in assays presented in Fig. 4C, and control cells were transfected with the empty IRES-GFP vector. GFP+ cells were sorted by FACS, chromatin was isolated, and ChIP was performed using an antibody that recognizes POU1F1. Following immunoprecipitation, qRT-PCR was used to measure the relative levels of POU1F1 binding in Pit-1/Triple cells that had been transfected with Nr4a2 plasmid (+Nr4a2), as well as those transfected with empty vector. Immunoprecipitations using IgG served as a negative control. *P < 0.05.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Isolation, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Negative Control

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: Immunoprecipitation of H3K27-acetylated (H3K27ac) chromatin in Pit-1/0 cells expressing Nr4a2 and/or Pou1f1. Pit-1/0 cells, which express neither Nr4a2 nor significant levels of Pou1f1, were transfected with Nr4a2-IRES-GFP, Pou1f1-IRES-GFP, or a 1:1 mixture of both expression vectors, and control cells were transfected with an empty IRES-GFP vector. Cells transfected with the 1:1 mixture of the Pou1f1 and Nr4a2 expression vectors express lower levels of each protein due to the concentration of each expression vector being halved during transfection to keep the total amount of DNA constant in each transfection. GFP+ cells were isolated by FACS and chromatin was isolated. ChIP was performed using an antibody that recognizes H3K27ac. Following ChIP, qRT-PCR was performed to assay the levels of H327 acetylation at the Gh promoter, the Prl promoter, and the MyoD promoter as a negative control. IgG controls (data not shown) were also included in this assay for all samples. In all cases, ChIP performed with IgG yielded <1% of input. *P < 0.05.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Immunoprecipitation, Expressing, Transfection, Control, Plasmid Preparation, Concentration Assay, Isolation, Quantitative RT-PCR, Negative Control

Journal: mBio

Article Title: Genome-Wide Profiling of Cervical RNA-Binding Proteins Identifies Human Papillomavirus Regulation of RNASEH2A Expression by Viral E7 and E2F1

doi: 10.1128/mBio.02687-18

Figure Lengend Snippet: RNASEH2A is regulated by viral E7 via E2F1 and its expression in association with E2F1 and PCNA in cervical and endocervical cancer and head and neck squamous cell carcinoma. (A) Read coverage maps showing the RNA expression levels of all 7 cervical cancer samples and 7 normal samples for RNASEH2A. Distributions of RNASEH2A reads (blue for normal, red for cancer) along with RNASEH2A transcript structure were visualized using the Integrative Genomics Viewer (IGV). (B) Correlation between RNASEH2A and HPV16 E7 mRNAs in 48 clinical cervical lesion samples, including 25 CIN2-CIN3 and 23 cervical cancer tissue samples. (C) siRNA-specific knockdown of HPV16 or HPV18 E7 expression in CaSki (HPV16 + ) cells or HPV18-immortalized human foreskin keratinocytes (HFK18) with respect to expression of RNASEH2A, PCNA, and NOVA1 was evaluated by Western blotting. si-NS, nonspecific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. Knockdown efficiency of E7 expression was evaluated by analysis of increased expression of p53 due to the E7 siRNA also targeting the overlapped bicistronic E6 RNA ; tubulin served as a sample loading control. (D) siRNA-specific knockdown of HPV16 or HPV18 E6 or E7 in CaSki (HPV16 + ) or HeLa (HPV18 + ) cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E6, HPV16 or HPV18 E6-specific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. (E and F) Specific-siRNA knockdown (E) or ectopic expression of E2F1 (F) in CaSki or HeLa cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E2F1, E2F1-specific siRNA; p, control vector; p-E2F1, E2F1-expression vector. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001. The relative expression levels of RNASEH2A in panels D, E, and F are shown as means ± SD for each treatment group determined in duplicate from three independent experiments, with the level in the si-NS (D and E) or vector control (F) group being set to 1. (G) Increased coexpression of RNASEH2A, PCNA, and E2F1 in cervical squamous cell carcinoma, endocervical adenocarcinoma (CESC), and head and neck squamous cell carcinoma (HNSC). Data were extracted from the TCGA data sets using TCGA2STAT R package version 1.2 ( https://CRAN.R-project.org/package=TCGA2STAT ).

Article Snippet: CaSki and HeLa cells were also transfected with 2 μg of an E2F1 expression vector (pRcCMV-HA-E2F1; Addgene, Watertown, MA) with X-tremeGENE HP DNA transfection reagent (Roche), and total RNA from the cells with E2F1 overexpression was analyzed for RNASEH2A expression by real-time RT-qPCR.

Techniques: Expressing, RNA Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Steady-state binding curves of monomeric Tspan12∆C, monomeric Fzd4, or heterodimeric Tspan12∆C/Fzd4∆C receptors in biotinylated nanodiscs binding to dimeric or ( B ) monomeric (C93A/C95A/C131A) Norrin by biolayer interferometry (BLI). Steady-state binding signal is plotted as a percent of B max for three independent replicates (mean ± SD). Affinities and kinetic constants are reported in . ( C ) Indicated concentrations of Norrin-1D4 dimer binding to Expi293 cells transfected with Fzd4, Tspan12, or both Fzd4 and Tspan12, detected with fluorescently labeled Rho1D4 antibody and quantified by flow cytometry. Mean ± SD of three independent experiments are plotted. Co-transfection of Tspan12 increased Norrin recruitment to Fzd4-transfected cells at 0.1, 0.32, 1, and 3.2 nM Norrin (two-tailed t-test p-values of 0.00026, 0.00079, 0.0049, and 0.0018, respectively). ( D ) β-Catenin pathway activation resulting from increasing concentrations of Norrin was assessed in Fzd1/2/4/5/7/8-knockout HEK293T cells transfected with Tspan12 siRNA or increasing amounts of Tspan12 plasmid, along with Fzd4 and TopFlash luciferase reporter plasmids. Data are plotted as mean ± SD from triplicate wells are representative of three independent experiments. Figure 4—source data 1. Interference shift, cell fluorescence, and luciferase activity values used to generate .

Article Snippet: Recombinant DNA reagent , Monomeric Norrin , This paper , RRID: Addgene_216386 , pAcGP67a vector; residues 33–133; MBP- and Rho-1D4-tagged; C93A/C95A/C131A; Deposited at Addgene (#216386).

Techniques: Binding Assay, Transfection, Labeling, Flow Cytometry, Cotransfection, Two Tailed Test, Activation Assay, Knock-Out, Plasmid Preparation, Luciferase, Fluorescence, Activity Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative biolayer interferometry (BLI) association and dissociation traces of dimeric Norrin binding to Fzd4 monomer or ( B ) Tspan12/Fzd4 heterodimer in nanodiscs. ( C ) Observed association rate constant K obs of Norrin dimer binding to Tspan12, Fzd4, or Tspan12/Fzd4 heterodimer in nanodiscs, plotted against Norrin concentration. Linear fits were used to obtain association rate constants reported in . Data represent mean ± SD for three independent replicates. ( D ) Representative BLI association and dissociation traces of monomeric Norrin (C93A/ C95A/C131A) binding to Fzd4 monomer or ( E ) Tspan12/Fzd4 heterodimer in nanodiscs. ( F ) Observed association rate constant K obs (mean ± SD) of Norrin monomer binding to Tspan12, Fzd4, or Tspan12/Fzd4 heterodimer in nanodiscs, plotted against Norrin monomer concentration. ( G ) Fzd4 surface expression on Expi293 cells transfected with empty vector, FLAG-Fzd4, or FLAG-Fzd4+Tspan12, which were then stained with M1 anti-FLAG antibody conjugated to Alexa Fluor 647. Cell fluorescence is measured by flow cytometry and plotted along with the median and interquartile range. Co-expression of Tspan12 modestly but significantly decreases surface expression of Fzd4 (Mann-Whitney test, p-value<0.0001 in each of three independent experiments).

Article Snippet: Recombinant DNA reagent , Monomeric Norrin , This paper , RRID: Addgene_216386 , pAcGP67a vector; residues 33–133; MBP- and Rho-1D4-tagged; C93A/C95A/C131A; Deposited at Addgene (#216386).

Techniques: Binding Assay, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence, Flow Cytometry, MANN-WHITNEY

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Analytical size exclusion traces of wild-type (WT) (dimeric) MBP-Norrin (yellow) and MBP-Norrin rendered monomeric (brown) via mutations C93A/C95A/C131A to eliminate the intermolecular disulfides. Purified protein was injected at 25 µM on a Superdex 200 Increase 10/300 column, resulting on an on-column concentration in excess of 2.5 µM assuming a 10-fold on-column dilution factor. ( B ) Non-reducing SDS-PAGE gel of WT and C93A/C95A/C131A MBP-Norrin. ( C ) Uranyl acetate negative stain micrograph of WT MBP-Norrin, prepared at 100 nM. Scale bar is 50 nm. Representative picked particles indicated in yellow. ( D ) Uranyl acetate negative stain micrograph of MBP-Norrin C93A/C95A/C131A, prepared at 100 nM. Scale bar is 50 nm. Representative picked particles indicated with circles. Yellow-circled particle appears to be large enough to potentially be a dimer; brown circles show some smaller species, which dominate. ( E ) 2D class averages of picked particles from C show two lobes, consistent with two copies of MBP-Norrin (54 kDa each). ( F ) 2D class averages of picked particles from D show small, single particles that are hard to align; they are about half the size of particles in E, consistent with one copy of MBP-Norrin. This suggests that MBP-Norrin C93A/C95A/C131A is monomeric at 100 nM. ( G ) β-Catenin transcriptional activity in response to 0.01–10 nM purified WT (dimeric) or 0.02–20 nM C93A/C95A/C131A (monomeric) Norrin, in Fzd1/2/4/5/7/8-knockout HEK293T cells transfected with Fzd4 and TopFlash luciferase reporter plasmids. Data are plotted as mean ± SD from n=3 replicate wells. Figure 4—figure supplement 4—source data 1. Original file of gel in . Figure 4—figure supplement 4—source data 2. Labeled gel in .

Article Snippet: Recombinant DNA reagent , Monomeric Norrin , This paper , RRID: Addgene_216386 , pAcGP67a vector; residues 33–133; MBP- and Rho-1D4-tagged; C93A/C95A/C131A; Deposited at Addgene (#216386).

Techniques: Purification, Injection, Concentration Assay, SDS Page, Staining, Activity Assay, Knock-Out, Transfection, Luciferase, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Hypothesis: Tspan12 could enhance Norrin signaling by enhancing interactions within the Norrin-LRP5/6-Fzd4-Dvl complex, including Fzd-Dvl binding and Norrin-LRP binding. ( B ) Representative biolayer interferometry (BLI) traces of the Dvl2 DEP domain associating to and dissociating from Fzd4 in nanodiscs containing 75:20:5 POPC:Ccholesterol:PIP 2 . ( C ) Equilibrium binding of the Dvl2 DEP domain to Fzd4 monomer or Tspan12/Fzd4 heterodimer in nanodiscs; affinities ± SEM are 183±24 and 279±46 nM, respectively. ( D ) Equilibrium binding of the Dvl2 DEP domain to Fzd4 monomer or Tspan12/Fzd4 heterodimer nanodiscs, each pre-saturated with 10 nM Norrin. Binding affinities are 161±21 and 274±39 nM (mean ± SEM), respectively, determined from three independent replicates. Affinities and kinetic constants are reported in . ( E ) The LRP6 E1E2 domain fully competes with Tspan12-Norrin binding, as shown by decreased equilibrium binding of 32 nM Norrin to Tspan12 immobilized on paramagnetic particles in the presence of increasing concentrations of purified LRP6 E1E2 domain. Norrin was quantified by western blot (anti-Rho1D4; see ) and plotted as a percent of bound Norrin in the absence of LRP6 E1E2. The curve was fit to a competitive binding model using known binding affinities of 10.4 nM for Tspan12-Norrin and starting concentrations of 50 nM Tspan12 and 32 nM Norrin; the best fit reported a Norrin-LRP6 E1E2 binding affinity of 1.06 µM (95% CI 0.747–1.51 µM). Data represent mean ± SD of three replicates. ( F ) β-Catenin transcriptional activity in response to no ligand, 1 nM recombinant Norrin, or Wnt3a conditioned media (Wnt3a CM) in Fzd1/2/4/5/7/8-knockout HEK293T cells transfected with Tspan12 siRNA or indicated amount of Tspan12_pTT5 plasmid, along with Fzd4 and TopFlash luciferase reporter plasmids. Data are plotted as mean ± SD from n=3 replicate wells. Figure 5—source data 1. Interference shift, band quantification, and luciferase activity values used to generate .

Article Snippet: Recombinant DNA reagent , Monomeric Norrin , This paper , RRID: Addgene_216386 , pAcGP67a vector; residues 33–133; MBP- and Rho-1D4-tagged; C93A/C95A/C131A; Deposited at Addgene (#216386).

Techniques: Binding Assay, Purification, Western Blot, Activity Assay, Recombinant, Knock-Out, Transfection, Plasmid Preparation, Luciferase

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Monomeric Norrin , This paper , RRID: Addgene_216386 , pAcGP67a vector; residues 33–133; MBP- and Rho-1D4-tagged; C93A/C95A/C131A; Deposited at Addgene (#216386).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Binding Assay, Expressing, Control, Reporter Assay, Software