Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Characterization of Esrrb-expressing cancer cell line. Esrrb status of two independent replicates of stable transfected control DU145-pc3.1 and DU145-Esrrb cells are tested by a quantitative PCR b Western blot and c reverse transcriptase PCR. a Relative mRNA concentrations of Esrrb were measured by qPCR, Esrrb transcripts concentration were determined by standard curve method and Esrrb concentration were first normalized to the concentration of house keeping gene GAPDH, then normalized to Esrrb/GAPDH ratio of DU145-pc3.1 cells. b Total protein was extracted form HEK293, DU145-Esrrb and control DU145-pc3.1 cells. Protein concentration of Esrrb was determined by western blot using GAPDH as internal control. c RT-PCR was performed on total RNA extracted from HEK293, DU145-esrrb and control DU145-pc3.1 cells. Esrrb was expressed in DU145-Esrrb cells, while Esrrg is not expressed in either DU145-pc3.1 and DU145-Esrrb cells

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Protein Concentration, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Esrrb-regulated mRNA validation. Left panel qPCR validation of Esrrb-regulated mRNAs. Expression values were firstly normalized to Gapdh and normalized ratios are further normalized to that of DU145-pc3.1. Error bars represent standard deviation. Student t test was performed for statistical analysis (*p < 0.05). Seven genes were differentially expressed in both RNA-seq and qPCR, 1 gene, TGFbeta, is not differentially expressed in either assay and serves as a negative control. Right panel RNA-Seq analysis result, fold change (FC) indicates the ratio of normalized read counts in DU145-Esrrb to that of DU145-pc3.1

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing, Standard Deviation, RNA Sequencing Assay, Negative Control

Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Transcriptome correlation and Esrrb altered mRNAs. a Transcriptome correlation analysis was performed using Spearman Ranking Correlation. Color represents the correlation coefficient. DY131 treatment to DU145-Esrrb cells results in the lowest correlation coefficient with DU145-pc3.1 cells. b Dot plot of Esrrb-induced gene expression alteration. Genes expressed at adequate level are tested for differential gene expression test. The plot was made by plotting the Log2FC (fold change) against the Log2 cpm (count-per-million) difference. Red color marks the genes that are significant differentially expressed (FDR < 0.05), and the blue lines marked the Log2FC cutoff value (Log2FC > 1 or Log2FC < −1). 67 genes passed both thresholds

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing

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