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

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