Journal: Cell death & disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma.

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Fig. 7 DSCR8 activates Wnt/β-catenin pathway by DSCR8/miR-485-5p/FZD7 axis. Rescue experiments revealed that DSCR8 negatively regulated the miR-485-5p expression (a, b), whereas positively regulated both the mRNA (a, b) and protein (c, d) expression of FZD7 by DSCR8/miR-485-5p/ FZD7 axis. c, d Western blot results from rescue experiments showed that DSCR8 positively regulated the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression by DSCR8/miR-485-5p/FZD7 axis. n = three repeats with similar results, *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing, Western Blot

Journal: Cell death & disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma.

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Fig. 9 Schematic of the proposed mechanism of DSCR8 in HCC. DSCR8 acts as a molecular sponge for miR-485-5p to regulate FZD7 expression, subsequently activating Wnt/β-catenin signal pathway to promote HCC progression

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing

Journal: The FASEB Journal

Article Title: Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families

doi: 10.1096/fj.201700144R

Figure Lengend Snippet: Constructs used in this study

Article Snippet: FZD7 , pCMV6-XL5 FZD7 , Origene , SC122259 , .

Techniques: Construct, Luciferase

Journal: Cell death & disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma.

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Fig. 7 DSCR8 activates Wnt/β-catenin pathway by DSCR8/miR-485-5p/FZD7 axis. Rescue experiments revealed that DSCR8 negatively regulated the miR-485-5p expression (a, b), whereas positively regulated both the mRNA (a, b) and protein (c, d) expression of FZD7 by DSCR8/miR-485-5p/ FZD7 axis. c, d Western blot results from rescue experiments showed that DSCR8 positively regulated the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression by DSCR8/miR-485-5p/FZD7 axis. n = three repeats with similar results, *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing, Western Blot

Journal: Cell death & disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma.

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Fig. 9 Schematic of the proposed mechanism of DSCR8 in HCC. DSCR8 acts as a molecular sponge for miR-485-5p to regulate FZD7 expression, subsequently activating Wnt/β-catenin signal pathway to promote HCC progression

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: a Data from bioinformatics tools (microRNA.org, TargetScan, and miRDB) showed that there were putative binding sites between 3′UTR of FZD7-wt and miR-485-5p. FZD7-mut means mutation of binding sites in 3′UTR of FZD7. b Luciferase reporter gene assays revealed that miR-485-5p negatively regulated the luciferase activity of FZD7-wt-3′UTR, rather than of DSCR8-mut-3′UTR. The mRNA ( c , d ) and protein ( e , f ) expression of FZD7 was negatively regulated by miR-485-5p. And FZD7 clone or siRNAs reversed the effects of miR-485-5p mimics or inhibitors on FZD7 expression ( c – f ). e , f Western blot results revealed that the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression were negatively regulated by miR-485-5p, while reversed by FZD7 clone and siRNA. n = three repeats with similar results, ** P < 0.01, *** P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Rescue experiments revealed that DSCR8 negatively regulated the miR-485-5p expression ( a , b ), whereas positively regulated both the mRNA ( a , b ) and protein ( c , d ) expression of FZD7 by DSCR8/miR-485-5p/FZD7 axis. c , d Western blot results from rescue experiments showed that DSCR8 positively regulated the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression by DSCR8/miR-485-5p/FZD7 axis. n = three repeats with similar results, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: DSCR8 acts as a molecular sponge for miR-485-5p to regulate FZD7 expression, subsequently activating Wnt/β-catenin signal pathway to promote HCC progression

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cachd1 is a novel Frizzled- and LRP6-interacting protein required for neurons to acquire left-right asymmetric character

doi: 10.1101/2022.05.16.492129

Figure Lengend Snippet: (A, left) Representative scatter plot of flow cytometry testing binding of FLAG-tagged CACHD1 prey protein to human FZD7-eGFP transiently transfected HEK293E cells (detected by phycoerythrin (PE)-conjugated secondary antibody; A, right) without (blue) or with (red) pre-incubation with anti-Frizzled antibody OMP-18R5; secondary only negative control (grey). n = 3; one-tailed paired t -test (D. F. = 2, t = 9.53, *** p = 0.0054). (B) Dot plot of human (blue diamonds) or zebrafish (blue circles) CACHD1, or negative control CD200R (grey) prey protein binding (∆M PE ) to cells transiently transfected with eGFP fusion protein constructs indicated (transfections verified by antibody labelling in bold). Each dot represents a single experiment; horizontal bars denote the mean and error bars represent 95% confidence intervals. One way Welch test of means (Cachd1 prey v. CD200R prey, not assuming equal variances; F = 132.32, D. F num = 30.00, D. F denom = 34.67, p = 5.09 × 10 −28 ), post hoc pairwise t -tests with non-pooled standard deviations, Benjamini & Hochberg correction for multiple testing; only significant differences between Cachd1 and CD200R prey for individual transfections are presented here for clarity, ** 0.05 > p > 0.01, *** 0.01 > p > 0.005, **** p < 0.005. (C) SPR-based determination of K D for mouse CACHD1 ECD analyte binding to immobilised FZD5 CRD , LRP6 P3E3P4E4 (3-4, left panel), and normalised response curves for different CACHD1 ECD :FZD CRD interactions. RU, response units.

Article Snippet: Mouse Fzd5 cysteine rich domain (UniProt: Q9EQD0, residues A27-T157), human FZD7 CRD (UniProt: O75084, residues Q33-G170), human FZD8 CRD (UniProt: Q9H461, residues A28-T158) were synthesized (Genscript).

Techniques: Flow Cytometry, Binding Assay, Transfection, Incubation, Negative Control, One-tailed Test, Protein Binding, Construct

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Activation with Wnt3a triggers the formation of a IFG-1r/Wnt3a/Fz7 complex. (A) Fz7 is enriched in GCPs. Western blot analysis of the distribution of the Wnt receptor Fz7 in different subcellular fractions of fetal brain: homogenate (H), low-speed supernatant (LSS), A-fraction (A), and growth cone particles (GCPs). Equal amounts of protein were loaded in all lines. Note the enrichment of Fz7 in GCPs around fourfold compared to (H). A blot showing the distribution of GAP-43, a protein highly enriched in GCPs, was included for comparison. (B , C) Wnt3a triggered the formation of a complex containing IGF-1r-Wnt3a-Fz7. Growth cone particles were incubated in control buffer or in the presence of 1.35 nM Wnt3a and immunoprecipitated with (B) Anti Wnt3a and probed with an antibody to the Wnt receptor Fz7 and (C) immunoprecipitated with β gc antibody to pull down IGF-1r and probed with anti Fz7. Immunoprecipitation of Wnt3a and β gc are shown to confirm successful IP and as a loading control. IP, immunoprecipitate; S, soluble fraction after immunoprecipitation. (D) Total protein from hippocampal neurons cultured for 20 h in the presence of 1.35 nm Wnt3a, deprived of any growth factor for 4 h, and stimulated (or not) with 1.35 nM Wnt3a for 5 min immunoprecipitated with anti β gc antibody to pull down the IGF-1r and probed with anti Fz7. Immunoprecipitation of β gc is shown to confirm successful IP and as a loading control. IP, immunoprecipitate; S, soluble fraction after immunoprecipitation. All blots are representative of at least three independent experiments.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Activation Assay, Western Blot, Comparison, Incubation, Control, Immunoprecipitation, Cell Culture

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Wnt3a polarizing effects required normal expression of IGF-1r. (A) Triple immunofluorescence micrographs of hippocampal neurons after 24 h in culture showing the distribution of the IGF-1r β gc subunit, a scrambled sequence RNA (GFP, top) or IGF-1r targeted shRNA (GFP, bottom), and the neuronal marker β-III tubulin (tub). Cells were cultured in medium containing 1.35 nM Wnt3a. The ssRNA transfected cells (top) showed a normal morphology. In contrast, the shRNA transfected cells (bottom) did not develop an axon. (B) Western blots showing protein levels of β gc, the Wnt receptor Fz7 (to control specificity, also serves as loading control) and cells transfected with a scrambled RNA sequence (ssRNA), and cells transfected with IGF-1r shRNA (shRNA). We observed a substantial decrease in β gc expression in the shRNA treated cells. (C) Percentage (±sem) of neurons at different specific stages of differentiation grown for 20 h in culture in control medium or in the presence of 1.35 nM Wnt3a and transfected with a scrambled sequence RNA (ssRNA) or IGF-1r targeted shRNA (shRNA) ( n = 3 independent experiments). At least 100 transfected cells (GFP positive) were scored for each condition. * p < 0.005 compared to ssRNA + Wnt3a. Scale bar: 20 μm. (D) Immunofluorescence of hippocampal neurons after 24 h of DIV showing the distribution of myc (marker of transfection with cDNA encoding for a constitutively active form of the PI3k catalytic subunit p110). GFP is a marker of transfection efficiency with the shRNA directed to IGF-1r.Calibration bar = 20 μm. (E) Percentage (±sem) of neurons at different specific stages of differentiation grown for 20 h in culture in the presence of 1.35 nM Wnt3a and transfected with IGF-1r shRNA (shRNA) or co-transfected with shRNA and a constitutively active form of the PI3k catalytic subunit p110 (p110CA). n = 3 independent experiments. Thirty or more transfected or co-transfected cells were scored in each experiment. * p < 0.02 compared to shRNA.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Expressing, Immunofluorescence, Sequencing, shRNA, Marker, Cell Culture, Transfection, Western Blot, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: (A) Wnt3a polarizing effect depends on the activation of Fz7. Double immunofluorescence micrographs of hippocampal neurons incubated for 24 h in the presence of 1.35 nM Wnt3a showing the distribution of the axonal marker Tau-1 and the neuronal marker β-III-tubulin. myc is a marker of transfection with the negative dominant form of Fz7-CRD. Scale bar: 20 mm. (B) Percentage (±sem) of neurons at different stages of differentiation grown for 20 h in medium containing 1.35 nM Wnt3a and transfected with pcDNA3.1-myc or Fz7-CRD ( n = 3 independent experiments). At least 20 transfected cells were scored for each condition. * p < 0.01 compared with pcDNA3.1-myc.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Activation Assay, Immunofluorescence, Incubation, Marker, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Wnt3a binds to Fz7 and cross-activate the IGF-1-PI3k-Akt pathway inducing initial axonal outgrowth and neuronal polarization. Challenging with 1.35 nM Wnt3a resulted in a polarized distribution of active (phosphorylated) IGF-1r and phosphorylated p85, the PI3k regulatory subunit, to one neurite of cells in stage 2 (see Figure ).

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: