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: 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: Oncotarget

Article Title: FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells

doi:

Figure Lengend Snippet: (A) Western blots of FOXM1 and the internal control β-ACTIN in whole cell lysates of A2780CP70 cells treated for 2 days with different concentrations of WNT3A and SFRP5 peptides. (B) Ad293 cells were transfected with the indicated concentrations of pcDNA3.1 (control vector) or pcDNA3.1 encoding SFRP5 and 500 ng pLuc-FOXM1 (FOXM1 promoter luciferase vector). The cells were harvested two days after transfection, and dual luciferase reporter assays were performed. FOXM1 promoter activity was expressed as the fold change relative to the control (pcDNA3.1). Each bars represents mean ± standard error of the mean from three independent experiments. **: P < 0.01; ***: P < 0.001. (C and D) Western blots of SFRP5, FOXM1, and the internal control β-ACTIN in Ad293 cells overexpressing a control vector (Vector) or shSFRP5 vector (shSFRP5) (C), and ES-2 and TOV-21G cells (D).

Article Snippet: Recombinant human SFRP5 and WNT3a peptides were purchased from R&D Systems.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay