Journal: eLife

Article Title: Anti-resonance in developmental signaling regulates cell fate decisions

doi: 10.7554/eLife.107794

Figure Lengend Snippet: ( A ) FACS data for Wnt I/O cell line with (+)/without (-) 24 hr of light exposure. Axes are of β-catenin (mCherry) v. TopFlash (APC-Cy5.5) ( B ) Live cell imaging of CRISPR tdmRuby3-β-cat, lentiviral 8X-TOPFlash-tdIRFP and DAPI, 16 hr after adding CHIR99021 (+CHIR) and Wnt3a (+Wnt3 a). ( C ) Quantifications of β-catenin and TopFlash fluorescence for no light, +16 hr light, +Wnt3 a, and +CHIR, where the mean of each condition was normalized to the mean of the no light condition and error bars represent SEM. ( D ) Flow for cell segmentation and heatmap generation from experimental data, using CellPose add TrackMate. From left to right: magenta (β-catenin) and cyan (TopFlash) images are passed into CellPose + Trackmate for segmentation and tracking. An example image of CellPose segmentation is shown under ‘segmented images,’ where the different colors correspond to the cell’s segmentation ID. Final images under ‘quantify and plot’ show quantification of β-catenin and TopFlash. ( E ) Mean fluorescent intensity (MFI) of β-catenin in the 24 hr light on condition, normalized to light off β-catenin. ( F ) MFI of TopFlash in the 24 hr light on condition, normalized to light off TopFlash. ( G ) Population mean MFI of β-catenin from live, single cell traces in indicated conditions, normalized to light off β-catenin. Initial drop in fluorescence is due to media bleaching. ( H ) Population mean MFI of TopFlash from live, single cell traces from indicated conditions, normalized to light off TopFlash. ( I ) Jitter plot of β-catenin mean nuclear fluorescent intensity (MFI) at the maximum intensity point in continuous light exposure conditions. Each point represents a single cell and the black line represents the mean of the population. ( J ) Jitter plot of TopFlash mean nuclear fluorescent intensity (MFI). Each point represents a single cell and the black line represents the mean of the population.

Article Snippet: HEK293T cells were treated using 10 μM of CHIR99201 (Stem Cell Technologies, 72052) or 2.5 nM of Wnt3a ligand (R&D Systems, 5036-WN) added into Dulbecco’s Modified Eagle Medium, high glucose GlutaMAX (Thermo Fisher Scientific, 10566016) medium supplemented with 10% fetal bovine serum (Atlas Biologicals, F-0500-D) and 1% penicillin-streptomycin.

Techniques: Live Cell Imaging, CRISPR, Fluorescence

Journal: Neoplasia (New York, N.Y.)

Article Title: An AXIN2 Mutant Allele Associated With Predisposition to Colorectal Neoplasia Has Context-Dependent Effects on AXIN2 Protein Function 1

doi: 10.1016/j.neo.2015.04.006

Figure Lengend Snippet: AXIN2- and trAXIN2-mediated inhibition of Wnt/β-catenin/TCF transcriptional targets is context dependent. (A) Transient ectopic expression of AXIN2 and trAXIN2 suppress Wnt3a-mediated TCF reporter gene activity. HEK293T cells were transiently transfected with the pCMV-3Tag vector, AXIN2 , or trAXIN2 expression constructs as well as TOPFlash reporter vector. Twenty-four hours after transfection, cells were treated with Wnt3a for 8 hours before harvesting for luciferase assays. Luciferase assays were performed in triplicate and mean and SDs are indicated. (B and C ) Stable expression of AXIN2 but not trAXIN2 inhibits Wnt3a-mediated induction of endogenous Wnt/β-catenin/TCF target genes in rat intestinal IEC-6 cells. IEC-6 cells were transduced with empty retroviral expression vector construct or constructs for AXIN2 or trAXIN2 , and drug selection was undertaken to create stable polyclonal cell lines. IB studies of the resultant IEC-6 cell lines show stable expression of AXIN2 or trAXIN2, as detected with anti-AXIN2 antibody (B). Stable IEC-6 transductants were treated for 16 hours with Wnt3a. The cells were then harvested, total RNA was collected, and expression of the indicated Wnt / β-catenin / TCF target genes was assessed in three separate quantitative RT-PCR experiments. The individual data points for three independent qPCR experiments with the mean of each group designated by a horizontal line are shown in C.

Article Snippet: Twenty-four hours after plating, the cells were treated with 200 ng/ml recombinant Wnt3a (ProSci, San Diego, CA) to induce target gene expression or with phosphate-buffered saline as a control.

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Transduction, Retroviral, Selection, Quantitative RT-PCR

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 1. miR‑214 is downregulated in liver cancer and targets Wnt3a. (A) Reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression of miR‑214 in 24 paired human hepatocellular carcinoma and non‑tumor tissues. (B) Relative expression of miR‑214 in liver cancer cell lines and a normal liver cell line. **P<0.01; *P<0.05. (C) miR‑214 seed region sequence in the 3'UTR of Wnt3a. (D) Wnt3a protein expression as detected by immunohistochemistry. (E) Protein expression levels of Wnt3a were measured by western blot analysis in HepG2 cells transfected with miR‑214 or miR‑ctrl. (F) miR‑214 was co‑transfected with pmirGLO, pmirGLO‑Wnt3a‑3'‑UTR‑wt or pmirGLO‑Wnt3a‑3'‑UTR‑mut in HepG2 cells. Relative luciferase activity was measured after 48 h. *P<0.05 vs. control. miR, microRNA; mut/M, mutant; UTR, untranslated region; wt/W, wild‑type.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Polymerase Chain Reaction, Expressing, Sequencing, Immunohistochemistry, Western Blot, Transfection, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 2. miR‑214 inhibits the proliferation of liver cancer cells. CCK8 assay was performed to detect the effects of miR‑214 on cell proliferation at 24, 48, and 72 h in (A) HepG2 and (B) Hep3B cells. CCK8 assay was performed to detect the effects of siWnt3a on cell proliferation at 24, 48 and 72 h in (C) HepG2 and (D) Hep3B cells. Wnt3a overexpression vector was co‑transfected with miR‑ctrl or miR‑214 into (E) HepG2 and (F) Hep3B cells, and cell proliferation was detected by CCK8 assay. *P<0.05; **P<0.01 vs. miR‑ctrl + Wnt3a‑ctrl. CCK8, Cell Counting kit‑8; ctrl, control; miR, microRNA; OD, optical density; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 3. Overexpression of miR‑214 or Wnt3a silencing affects cell cycle progression. Cell cycle analysis of (A) HepG2 and (B) Hep3B cells following transfection with miR‑214 or miR‑ctrl for 48 h. Cell cycle analysis of (C) HepG2 and (D) Hep3B cells following transfection with siWnt3a or si‑ctrl for 48 h. *P<0.05. ctrl, control; miR, microRNA; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Over Expression, Cell Cycle Assay, Transfection, Control, Small Interfering RNA

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) Schematic of the protocol for assaying the correlation between Wnt3a signaling and glucose metabolism in the brain. After the indicated treatments, cognitive tests related to hippocampal function were performed: Large open field (B), NOR (C), NOL (D) and memory flexibility (E). Data obtained from APP/PS1 mice (under blue line). Data represent the mean ± SEM of n = 9 (number of animals), *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques:

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) Aβ oligomer structure determined by electron microscopy, treatment with Aβ reduced cell viability in a time-dependent manner, and the decrease in cell viability induced by Aβ was partially blocked by co-incubation with Wnt3a (arrow indicates apoptotic nucleus), n:3. (B) In contrast, co-incubation with Wnt3a+Aβ and Cyt B diminished the neuroprotective effect of the Wnt ligand. (C) Incubation with Aβ induced a dramatic decrease in 2-DG uptake. Wnt3a blocked this decrease, and Dkk partially inhibited the protective effect of Wnt3a. (D) The treatment of hippocampal neurons with ANDRO and Li stimulated the uptake of 2DG and this is inhibited by Cyt B. Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: Electron Microscopy, Incubation

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: The effects of the indicated treatments on the brain mRNA levels of genes encoding several proteins involved in glucose metabolism, including Campkiv (A), Cyclin D1 (B), c-Myc (C), which are known target genes of Wnt3a signaling. These target genes were increased by ANDRO and Li. The effect of treatments on the mRNA levels encoding additional metabolic proteins: Hk (D), Pk1 (E), Pfk1 (F), Akt (G) and Ampk (H). The mRNA levels of Hk, Pk1, Pfk1 and Akt increased after treatment with ANDRO. While the treatment with Li just recover the mRNA levels of Hk, Pkf1. Data from APP/PS1 mice (under blue line). Data represent the mean ± SEM of n = 3 (samples obtained from 3 different animals, by treatment), each performed in triplicate, *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques:

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) A time course of treatments showing that Aβ decreases the glycolytic rate in hippocampal neurons, in vitro. This effect was abolished by co-incubation with Wnt3a and this restoration was blocked by Dkk. (B) The activity of HK after 12 h of treatment with Aβ and/or the indicated compounds. (C) The activity of the PPP pathway after treatment with Aβ and the indicated compounds. This pathway remained unaltered. (D) ATP levels after Aβ treatment were dramatically reduced but rescued by co-treatment with Wnt3a. (E) The decrease in the levels of ATP correlated with the ATP/ADP ratio. Both ATP (E) and the ATP/ADP ratio increased in the presence of Wnt3a. Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: In Vitro, Incubation, Activity Assay

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) The uptake of radioactive glucose in slices obtained from Wt and APP/PS1 mice (under blue line). Slices were treated with the indicated drugs for 1 h and then glucose uptake was measured. Treatment with Wnt3a and Wnt agonists increased the uptake of glucose in APP/PS1 slices and this was blocked by oligomycin. (B) The glycolytic rate after treatment with the indicated drugs. The decreased glycolytic rate of APP/PS1 slices was rescued by Wnt signaling. The activity of two key regulatory glycolytic enzymes, HK and PFK (C and D, respectively). The treatments, with the exception of rWnt3a affecting PFK, did not rescue the APP/PS1 mediated decreases in HK or PFK activity. Both ATP and the ATP/ADP ratio were decreased in slices from APP/PS1 mice, and both were increased in the presence of the agonists of Wnt3a signaling (E and F, respectively). Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

doi: 10.1186/s12964-020-00559-0

Figure Lengend Snippet: Inducible RNF43 and RNF43ΔPA expression in T-REx 293 cell line. a. Schematic representation of the used experimental models. T-REx 293 cell lines inducibly expressing tagged wild type RNF43 or its variant lacking PA (aa 87–186) domain. Supplementation of culture medium with Tet drives transgene expression. b. Western blot showing Tet-induced expression of HA-tagged RNF43 constructs in stable sell lines. β-actin served as loading control. c. Expression of RNF43 (c) , ZNRF3 (c’) and CTNNB1 (c”) , genes in response to the 80 ng/ml rWNT3A, 50 ng/ml rRSPO1 and tetracycline overnight treatments in the parental, RNF43 and RNF43ΔPA (clone #1 and clone #2) TetON cell lines assessed by the qPCR. Data are presented as relative expression, 2 −ΔΔCt ± SD. d. Immunofluorescence of RNF43 and RNF43ΔPA intracellular localization. GFP with plasma membrane targeting signal served as plasma membrane marker (in green), HA tag for the detection of exogenous RNF43 and RNF43ΔPA mutant (in red). Nuclei stained with TO-PRO-3 Iodide are shown in blue. Scale bars represent 25 μm. Signals intensities were measured along selections and plotted. e. T-REx 293 cells overexpressing RNF43 were compared with the parental cell line for their ability to mediate TCF/LEF-dependent transcription in the Top flash dual luciferase assay. Response to 80 ng/ml rWNT3A, 25 ng/ml rRSPO1 and Tet treatments was measured after overnight incubation. Values are normalized to the unstimulated control cells. N = 4, unpaired two-tailed t-test ** p < 0.01, *** p < 0.001. f. Western blot analysis of canonical Wnt pathway activation by application of the increasing concentrations of rWNT3A (40, 60 and 100 ng/ml) after 3 h of treatment. In cells overexpressing RNF43 phosphorylation of S1490-LRP6 and DVL2 (arrowheads) were weaker. Signal corresponding to the β-actin signal was used as loading control and HA tag specific antibody for RNF43 detection, N = 3

Article Snippet: For the purpose of canonical Wnt pathway stimulation, cells were treated with the recombinant human WNT3A (rWNT3A) (CF 5036-WN-CF, RnD Systems) for 3 h or overnight for the Top flash dual luciferase assay in the indicated concentrations (40–100 ng/ml).

Techniques: Expressing, Variant Assay, Western Blot, Construct, Control, Immunofluorescence, Clinical Proteomics, Membrane, Marker, Mutagenesis, Staining, Luciferase, Incubation, Two Tailed Test, Activation Assay, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

doi: 10.1186/s12964-020-00559-0

Figure Lengend Snippet: RNF43ΔPA inhibits canonical Wnt signaling pathway. a . Top Flash dual luciferase assay performed in the T-REx 293, RNF43 TetON and two RNF43ΔPA TetON cell lines in the response to the 100 ng/ml rWNT3A and 50 ng/ml rRSPO1 treatments in the presence of 0.5 μM LGK-974. Both RNF43 variants efficiently inhibited cellular responses. Results were normalized to Tet free conditions. N = 4, unpaired two-tailed t-test *** p < 0.001, **** p < 0.0001. b. Expression of the canonical Wnt signaling target gene AXIN2 in response to the rWNT3A (100 ng/ml) and rRSPO1 (50 ng/ml) treatments. Cells overexpressing RNF43ΔPA mutant, similarly to the wild type RNF43, showed weaker AXIN2 expression. Results were normalized to the assay values of the unstimulated samples. N = 3, unpaired two-tailed t-test. * p < 0.05, ** p < 0.01, *** < 0.001. Data are presented as 2 −ΔΔCt ± SD. c. Schematic representation of performed experiments elucidating an impact of RNF43ΔPA on the different canonical Wnt signaling modes of activity – Wnt pathway activation and RNF43 constructs expression simultaneously, before and after pathway stimulation. d. Western blot analysis of RNF43ΔPA impact on the canonical Wnt pathway components. Phosphorylation of S1490 of LRP6 together with the DVL2 and DVL3 activation manifested by electrophoretic, phosphorylation specific shifts (arrowheads) upon RNF43 or RNF43ΔPA tetracycline forced expression along the 80 ng/ml rWNT3A and 25 ng/ml rRSPO1 treatments (d), before them (d’) or after (d”) stimulations. Both variants of RNF43 showed inhibitory effect on the β-catenin dependent Wnt signaling in the all tested conditions. LGK-974 was used to inhibit autocrine production of the Wnt ligands, HA tag signal corresponds to the RNF43 proteins and β-actin was employed as loading control

Article Snippet: For the purpose of canonical Wnt pathway stimulation, cells were treated with the recombinant human WNT3A (rWNT3A) (CF 5036-WN-CF, RnD Systems) for 3 h or overnight for the Top flash dual luciferase assay in the indicated concentrations (40–100 ng/ml).

Techniques: Luciferase, Two Tailed Test, Expressing, Mutagenesis, Activity Assay, Activation Assay, Construct, Western Blot, Phospho-proteomics, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

doi: 10.1186/s12964-020-00559-0

Figure Lengend Snippet: Preparation of cells lacking functional RNF43/ZNRF3 proteins. a. In order to generate double knockout lines, CRISPR/Cas9 method was applied to introduce mutations in the RNF43 and ZNRF3 genes of T-REx 293 cells. Sequencing results of loci targeted by the CRISPR/Cas9 approach. PAM sequences are underlined. Modifications effects on the protein sequence were predicted. b. RNF43 / ZNRF3 dKO cells had higher β-catenin dependent transcriptional activity in response to the overnight incubation with different rWNT3A doses (40, 60 and 80 ng/ml). Also, dKO cell line was insensitive to the 25 ng/ml rRSPO1 treatment. All values are normalized to the unstimulated control cells results. N = 5 (wild type cells), N = 3 (dKO), unpaired two-tailed t-test * p < 0.05, ** p < 0.01, *** < 0.001. c. Western blot analysis of canonical Wnt pathway activation in response to the increasing doses of rWNT3A (40, 60 and 100 ng/ml) after 3 h long treatments. RNF43 / ZNRF3 dKO exhibited stronger response to the rWNT3A in comparison to the parental cells and rRSPO1 stimulation had no effect. Signal corresponding to the β-actin signal was used as loading control, N = 3

Article Snippet: For the purpose of canonical Wnt pathway stimulation, cells were treated with the recombinant human WNT3A (rWNT3A) (CF 5036-WN-CF, RnD Systems) for 3 h or overnight for the Top flash dual luciferase assay in the indicated concentrations (40–100 ng/ml).

Techniques: Functional Assay, Double Knockout, CRISPR, Introduce, Sequencing, Activity Assay, Incubation, Control, Two Tailed Test, Western Blot, Activation Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

doi: 10.1186/s12964-020-00559-0

Figure Lengend Snippet: Both RNF43 and RNF43ΔPA rescue phenotypes of RNF43/ZNRF3 dKOs cells. a. pcDNA4 plasmids encoding RNF43 wt, RNF43ΔPA and enzymatically inactive RNF43 Mut1 were transfected into CRISPR/Cas9 derived RNF43 / ZNRF3 dKO #1 and #2 T-Rex cell lines. Monoclonal stable TetON cell lines were derived thanks to antibiotic selection and colonies picking. b., c. Top flash dual luciferase assay in the RNF43 / ZNRF3 dKO #1 ( b ) and #2 ( c ) derivates expressing RNF43 wt, RNF43ΔPA or RNF43 Mut1 in the tetracycline sensitive way. Cells in all conditions were treated with the LGK-974. Tetracycline induced expression of RNF43 and its variants, recombinant WNT3A (80 ng/ml) activated canonical Wnt signaling and rRSPO1 (100 ng/ml) co-treatment was used to antagonize RNF43 action. Results were normalized to the untreated samples values and compared using unpaired t-test * p < 0.05, ** p < 0.01 N = 3 or 4. d., d’., d”., d”’, e., e’., e”. e”’. Top flash samples were also analyzed by the Western blot for further investigation of the Wnt signaling activity in presence of different RNF43 variants in the RNF43 / ZNRF3 dKO #1 ( d ) and dKO #2 ( e ). Tetracycline forced expression of both RNF43 wt and RNF43 RNF43ΔPA, but not RNF43 Mut1, suppressed LRP6 1490S, DVL2 and DVL3 phosphorylation events. Recombinant RSPO1 treatment antagonized RNF43 wt, but not RNF43 lacking PA domain. RNF43 lacking enzymatic activity (Mut1) had no effect on the canonical Wnt signaling. DVL2 (d’, e’), DVL3 (d”, e”) and LRP6 (d”’, e”’) activation was quantified by the ImageJ software and presented as ratios of phosphorylation specific upper band to the lower one (arrowheads). Results were normalized to the untreated samples values and compared by t-test, N = 3 * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: For the purpose of canonical Wnt pathway stimulation, cells were treated with the recombinant human WNT3A (rWNT3A) (CF 5036-WN-CF, RnD Systems) for 3 h or overnight for the Top flash dual luciferase assay in the indicated concentrations (40–100 ng/ml).

Techniques: Transfection, CRISPR, Derivative Assay, Selection, Luciferase, Expressing, Recombinant, Western Blot, Activity Assay, Phospho-proteomics, Activation Assay, Software

Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Protein-Protein interactions, Cell Culture