Journal: bioRxiv

Article Title: Applying the auxin-inducible degradation (AID) system for rapid protein depletion in mammalian cells

doi: 10.1101/182840

Figure Lengend Snippet: A) Schematic of Cas9 in complex with a sgRNA, targeted to its complementary DNA sequence. Relative locations of the PAM motif (shown in orange), and nuclease cleavage sites are indicated. B) Diagram of oligonucleotide design for sgRNA cloning into PX459. A 20-nt oligonucleotide sequence is chosen, that targets the desired genomic sequence. If this sequence does not begin with a “G”, then a G should be appended to maximize expression from the U6 promoter (shown underlined). Overhangs must then be added to the oligos, with sequences complementary to those left by BsmBI digest of the PX459 vector. Oligos are then resuspended, annealed, and phosphorylated, after which they are ready for ligation into BsmBI-digested PX459.

Article Snippet: Materials required Expression plasmid for OsTIR1 pBabe Neo osTIR1-9Myc (Addgene #80072) pBabe Blast osTIR1-9Myc (Addgene #80073) pBabe Puro osTIR1-9Myc (Addgene #80074) px459 plasmid encoding sgRNA targeting desired genomic locus PX459 V2.0 (Addgene #62988) PX459 VQR (Addgene #101715) PX459 VRER (Addgene #101716) PX459 EQR (Addgene #101732) PX458 (Addgene #48138) PX458 VQR (Addgene #101727) PX458 VRER (Addgene #101728) PX458 EQR (Addgene #101731) PX330 (Addgene # 42230) PX330 VQR (Addgene #101730) PX330 VRER (Addgene #101729) PX330 EQR (Addgene #101733) Plasmid template containing the mini AID tag pcDNA5/FRT miniAID-EGFP (Addgene #101713) pcDNA5/FRT EGFP-miniAID (Addgene #101714) Cells of choice for validation experiments and growth media for those cells Antibodies specific to the epitope tag of the OsTIR1 (i.e., Myc) Standard immunofluorescence and immunoblotting reagents PCR purification/concentration kit Transfection or nucleofection reagents 1.1 Recipes Polyethylenimine (PEI) (1 mg/mL) Dissolve PEI powder (25 kDa, linear) to a concentration of 1 mg/ml in water which has been heated to 80°C Allow solution to cool to room temp.

Techniques: Sequencing, Cloning, Expressing, Plasmid Preparation, Ligation

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Generation of HAP1 SGFP2-CTNNB1 cell lines. A) Cartoon depicting the current model of the WNT/CTNNB1 pathway. In the absence of WNT ligands (left, “OFF”), free cytoplasmic CTNNB1 is captured by the destruction complex consisting of AXIN, APC, CSNK1 and GSK3, which leads to its phosphorylation, BTRC mediated ubiquitination and subsequent proteasomal degradation, resulting in low levels of CTNNB1 in the cytoplasm and nucleus. Binding of the WNT protein (right, “ON”) to the FZD and LRP receptors inhibits the destruction complex through DVL. CTNNB1 accumulates in the cytoplasm and subsequently translocates to the nucleus, where it promotes the transcription of target genes, such as AXIN2, as a co-activator of TCF/LEF transcription factors. B) Cartoon depicting exon 2 of the CTNNB1 locus, which contains the start codon, and the CTNNB1 protein before (top) and after (bottom) introduction of the SGFP2 by CRISPR/Cas9 mediated homology directed repair. C) Schematic of the experimental workflow and timeline for generating HAP1 SGFP2-CTNNB1 clones. Cas9, gRNA and repair templates are transfected as plasmids. The repair template contains the coding sequence of SGFP2 surrounded by 800 bp homology arms on either side and lacks the gRNA recognition site (see supplement 2 of this figure). A short puromycin selection step is included from 24-48 hours after transfection to enrich for transfected cells. Haploid, GFP-positive cells are sorted and single cell clones are expanded for further analysis. D-F) FACS plots illustrating control (D) and SGFP2-CTNNB1 tagged cells (E-F). D) Cells transfected with Cas9 and gRNA in the absence of a repair template were used to set the gate for SGFP2-positive events. E) A small population of cells expressing low levels of SGFP2 can be detected when cells are transfected with Cas9, gRNA and repair template. F) Treatment for 24 hours of cells similar to those depicted in (D) with 8μM CHIR99021 does not change the amount of cells that are SGFP2 positive, but increases the SFP2 signal, most likely reflecting an increase in SGFP2-tagged beta catenin levels on a per cell basis and supporting the notion that the gated events indeed represent successfully tagged cells. The following figure supplements are available for : : FACS Gating strategy for haploid HAP1 cells. : SGFP2-CTNNB1 locus.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Binding Assay, CRISPR, Clone Assay, Transfection, Sequencing, Selection, Expressing

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: SGFP2-CTNNB1 locus A) Detailed view of CTNNB1 exon 2 depicting gRNA design relative to the wildtype (top) and repaired (bottom) CTNNB1 allele. Note that the repair template contains the same sequence as the repaired allele depicted here. CTNNB1 sequences are shown in capital letters, SGFP2 sequences shown in lowercase. 5’ UTR, SGFP2 and CTNNB1 and intron regions are indicated below the colored boxes. The gRNA (white arrow box above sequence) overlaps the start codon (depicted in bold), resulting in a Cas9-mediated double-strand break in the 5’UTR (predicted cut site indicated by dotted line and scissor, PAM site underlined). After successful homologous recombination, most of the gRNA binding site is destroyed, thus minimizing the chance of cutting the repair template or re-cutting the repaired allele. PCR based screening confirmed that 22/23 single-cell sorted clones indeed showed an integration of the expected size at the CTNNB1 locus. Complete sequence coverage of the insertion site in exon 2 was obtained for 9/11 sequenced clones, of which 8 showed the desired repair, and 1 clone showed an additional point mutation in the repaired locus. B-C) Sequencing of three independent HAP1 SGFP2-CTNNB1 clones on the 5’ (B) and the 3’ (C) end of SGFP2 integration in exon 2 of CTNNB1 . Sanger sequencing of the endogenous CTNNB1 locus of clone 1, clone 2 and clone 3 shows an exact match to the design and thus correct homology directed repair.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Sequencing, Homologous Recombination, Binding Assay, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Functional validation of three independent HAP1 SGFP2-CTNNB1 clones. A) Western blot, showing CTNNB1 (HAP1 WT ) and SGFP2-CTNNB1 (HAP1 SGFP2-CTNNB1 clone 1, 2 and 3) accumulation in response to CHIR99021 treatment. All panels are from one blot that was cut at the 70 kDa mark and was stained with secondary antibodies with different fluorophores for detection. Top: HAP1 WT cells express CTNNB1 at the expected wild-type size. Each of the three clonal HAP1 SGFP2-CTNNB1 cell lines only express the larger, SGFP2-tagged form of CTNNB1, that runs at the expected height (∼27 kDa above the wild-type CTNNB1). Middle: Only the tagged clones express the SGFP2-CTNNB1 fusion protein, as detected with an anti-GFP antibody at the same height. Bottom: alpha-Tubulin (TUBA) loading control. A representative image of n=3 independent experiments is shown. B) Quantification of Western blots from n=3 independent experiments, including the one in (A), confirming that the accumulation of CTNNB1 in response to WNT/CTNNB1 pathway activation is comparable between HAP1 WT and HAP1 SGFP2-CTNNB1 cells. Horizontal bar indicates the mean. C) Graph depicting the results from a MegaTopflash dual luciferase reporter assay, showing comparable levels of TCF/LEF reporter gene activation for HAP1 WT and HAP1 SGFP2-CTNNB1 cells in response to CHIR99021 treatment. Data points from n=3 independent experiments are shown. Horizontal bar indicates the mean. Values are depicted relative to the DMSO control, which was set to 1 for each individual cell line. D) Graph depicting AXIN2 mRNA induction in response to CHIR99021 treatment, demonstrating that induced expression of an endogenous target gene is comparable between HAP1 WT and HAP1 SGFP2-CTNNB1 cells. Data points represent n=3 independent experiments. Horizontal bar represents the mean. HPRT was used as a reference gene. Values are depicted relative to the HAP1 WT DMSO control, which was set to 1. E) Representative confocal microscopy images of the three HAP1 SGFP2-CTNNB1 clones after 4-hour vehicle control or 100ng/ml WNT3A treatment from n=1 biological experiment, revealing intracellular accumulation of SGFP2-CTNNB1 (green). Nuclei were counterstained with SiR-DNA dye (magenta). Scale bar is 10μm. The following figure supplements are available for : : Verification of the WNT/CTNNB1 responsiveness of HAP1 cells.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Functional Assay, Clone Assay, Western Blot, Staining, Activation Assay, Luciferase, Reporter Assay, Expressing, Confocal Microscopy

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Verification of the WNT/CTNNB1 responsiveness of HAP1 cells. A) Graph depicting AXIN2 qRT-PCR results from HAP1 WT cells treated with the indicated range of CHIR99021 (1-10 µM) or DMSO vehicle control (0 µM) for 24 hours. HPRT was used as a reference gene. Error bars represent standard deviation within technical triplicates from n=1 biological experiment. Based on this, we selected 4uM and 8 µM as intermediate and high levels of WNT/CTNNB1 pathway induction for follow up experiments. B) Western blot, showing the increase in total (top) and non-phosphorylated (i.e. active) CTNNB1 levels (middle) in response to pathway stimulation. HAP1 WT cells were treated for 24 hours with 4 or 8 µM CHIR99021, or DMSO vehicle control (0 µM). Alpha-Tubulin (TUBA, bottom) serves as a loading control. C-D) Quantification of the western blot from (B) depicting the relative fold change of total CTNNB1 (C) or non-phosphorylated CTNNB1 (D) to DMSO control corrected for Tubulin loading. E) Representative confocal microscopy images of three independent HAP1 SGFP2-CTNNB1 clones, treated for 24 hours with 4 or 8 µM CHIR99021, or DMSO vehicle control. Scalebar is 10 µm.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Quantitative RT-PCR, Standard Deviation, Western Blot, Confocal Microscopy, Clone Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Live imaging of HAP1SGFP2-CTNNB1. A) Representative stills from confocal time-lapse experiments corresponding to Supplementary Movies 1-2, showing an increase of SGFP2-CTNNB1 after treatment with 100 ng/ml WNT3A (bottom) relative to a vehicle control (BSA) treated sample (top). Scale bar = 20 μm. B-D) Quantification of time-lapse microscopy series, using the segmentation pipeline shown in (E). Arrow indicates the moment of starting the different treatments (T, see legend in B for details). B-C) Graph depicting the normalized intensity of SGFP2-CTNNB1 in the cytoplasm (B) or nucleus (C) over time. Solid lines represent the mean normalized fluorescence intensity and shading indicates the 95% confidence interval. n=155-393 cells for each condition and time point, pooled data from n=3 independent biological experiments. D) Graph depicting the nuclear/cytoplasmic ratio of SGFP2-CTNNB1 over time, calculated from raw intensity values underlying (B) and (C). E) Segmentation of nuclei (top) and cytoplasm (bottom) based in the SiR-DNA signal and SGFP2-CTNNB1 signal. Scale bar = 20μm. The following figure supplements are available for : : Verification of imaging results with WNT3A three independent HAP1 SGFP2-CTNNB1 clones. : Difference analysis of SGFP2-CTNNB1 fluorescence. : Unnormalized nuclear and cytoplasmic intensity measurements

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Imaging, Time-lapse Microscopy, Fluorescence, Clone Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Difference analysis of SGFP2-CTNNB1 fluorescence. A-B) Plots depicting the relative intensity (left) and the difference in relative intensity to BSA treated cells (right) in the cytoplasm (A) and nucleus (B) after 4 hours of treatment. Circles indicate the median value and bars indicate the 95% CI. In the relative intensity plot (left) the distribution is built from individual data points in a violin-type fashion to faithfully represent the distribution of data. In the difference plot (right) the distribution of differences is represented in a half violin plot. If the 95% CI in the difference plot does not overlap the zero line, which indicates no difference, the sample is significantly different from BSA control condition. C-D) Plots depicting the difference in relative intensity in the cytoplasm (C) and nucleus (D) between the moment of addition and 1 hour of treatment. Titles indicate the time (hh:mm). The distribution of differences is represented in a half violin plot. Circles indicate the median value and bars indicate the 95% CI. If the 95% CI does not overlap the zero line, which indicates no difference, the sample is significantly different from the BSA control condition.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Graphs showing quantification of time-lapse microscopy experiments with three independent HAP1 SGFP2-CTNNB1 clones. Stills of this experiment are shown in . Segmentation was performed as described in . Arrow indicates the moment of starting the different treatments (BSA in red or 100 ng/ml WNT3A in blue). Solid lines represent the mean normalized intensity and shading the 95% confidence interval in the cytoplasm (A) or nucleus (B). Line pattern indicates the three different clones. n=13-158 cells for each condition and time point for n=1 biological experiment.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Time-lapse Microscopy, Clone Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Two diffusion-component fit-model for SGFP2-CTNNB1 FCS measurements. A) Schematic representation of the point FCS technique, depicting the confocal volume with fluorescent particles diffusing in and out. Particles in FCS are defined by their coherent movement; therefore, a particle can be made up of monomers or multimers in isolation or complexed to unlabeled molecules. B) Schematic representation of intensity fluctuations over time as measured in the confocal volume. Fluctuations are the result of both photo-physics (e.g. blinking of the fluorophore) and diffusion. C) Graphical representation of the two diffusion-component fitting model used for our autocorrelation curves. T trip describes the blinking of the SGFP2 fluorophore and the after-pulsing artefact. T diff1 and T diff2 describe the monomeric and complexed form of SGFP2-CTNNB1, respectively. Details of all fitting parameters are described in Materials and Methods. D) Representative confocal images of HAP1 SGFP2-CTNNB1 cells treated for 4 hours with BSA (left) or 100 ng/ml WNT3A (right). E) Zoom in of the white rectangle in (D), with representative locations of FCS measurement points for cytoplasm (C) and nucleus (N) indicated with white crosses in the SGFP2-CTNNB1 channel and transmission channel. F-G) Fitting of a representative autocorrelation curve with one unfixed diffusion-component (F) or a two diffusion-component model (G), where the first diffusion component was fixed to the speed of free monomeric SGFP2-CTNNB1 (14.9µm 2 /s) and the second diffusion component was unfixed. The black line represents the autocorrelation curve generated from the FCS measurement, the red line represents the fitted model. The residuals after fitting of 25 individual curves are shown in the upper right corner of the graphs.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Diffusion-based Assay, Isolation, Transmission Assay, Generated

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Abundance and mobility of SGFP2-CTNNB1 molecules in living cells after 4 hours WNT3A treatment or control. Details on sample size and statistics can be found in supplementary file 1. A) Graph depicting the total concentration of SGFP2-CTNNB1 particles (monomeric plus complexed) as measured with FCS. B) Graph depicting the concentration of SGFP2-CTNNB1 particles with the fast diffusion component (i.e. free monomeric). C) Graph depicting the concentration of SGFP2-CTNNB1 containing particles with the slow diffusion component (i.e. complex associated).D-E) Graphs depicting the fraction (D) and speed (E) of the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS. F) Example of typical regions of interest in two cells used in N&B analysis. Solid line represents the analysis ROI, dashed line, marks the outline of the nuclear envelope. G) Schematic representation of a confocal volume with different brightness species. On the left are 6 monomers with a brightness of 1, on the right 2 trimers with a brightness of 3, both result in a fluorescence of 6. N&B analysis is able to extract the number and the brightness of such samples, for more detail see supplement 1 of this figure. H) Graph depicting the molecular brightness of SGFP2-CTNNB1 in the cytoplasm and nucleus relative to controls as measured with N&B in the same subcellular compartments. EGFP monomer was used for normalization and EGFP dimer as a control for N&B measurements. The following supplements are available for : : Quantification of SGFP2-CTNNB1 particles, fluorescence and fluorescence lifetime. : Number and Brightness analysis Supplementary File 1: Lists all summary statistics (mean, median, 95% confidence intervals, differences, p-values) of the FCS and N&B parameters show in and Figure 6 supplement 1.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Concentration Assay, Diffusion-based Assay, Fluorescence

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Quantification of SGFP2-CTNNB1 particles, fluorescence and fluorescence lifetime. Details on sample size and statistics can be found in supplementary file 1. A) Graph depicting the total number of SGFP2-CTNNB1 particles as measured with N&B. B) Graph depicting the average fluorescence intensity at the start of the FCS measurement. The increase in SGFP2-CTNNB1 fluorescence in the cytoplasm (2.1-fold) exceeds the increase in the SGFP2-CTNNB1 concentration (1.1-fold, ), but does correspond to the relative increase measured by time-lapse imaging (1.7-fold, ). C) Graph depicting fluorescence lifetimes calculated from the FCS measurements. The Fluorescence lifetime of SGFP2-CTNNB1 is independent of the subcellular compartment and treatment. Therefore, photophysical effects are not the cause for the difference between the fold-change in fluorescence and concentrations of the FCS measurements as described in (B).

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Fluorescence, Concentration Assay, Imaging

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Computational model of WNT/CTNNB1 based on FCS concentrations for free and complexed CTNNB1 ( - ). A) Schematic overview of the model. DC=destruction complex, DC*= DVL-inactivated DC, CB=cytoplasmic CTNNB1, CB*=phosphorylated CB, NB=nuclear CTNNB1, TCF=TCF/LEF transcription factors, DVL=WNT-activated DVL. The model assumes that there is no activated DVL in the absence of WNT, therefore k5/k4 do not play any role in the WNT ‘OFF’ equilibrium. Note that CB* is degraded and therefore plays no role in the model. B) Graph depicting the modelled concentrations of cytoplasmic components over time. The black line indicates total concentration of cytoplasmic CTNNB1, corresponding to . C) Graph depicting the modelled concentrations of nuclear components over time. The black line indicates total concentration of nuclear CTNNB1, corresponding to . D) Graph depicting the ratio of total nuclear and cytoplasmic CTNNB1 over time, corresponding to the measurements in 4E. E) Graph depicting the DC-bound CTNNB1 fraction ratio over time. F) Graph depicting the TCF-bound CTNNB1 fraction ratio over time.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Generation and characterization of a S45F mutant cell line (HAP1 SGFP2-CTNNB1(S45F) ). A-C) FACS graphs showing the selection strategy for HAP1 SGFP2-CTNNB1(S45F) cells. HAP1 SGFP2-CTNNB1 clone 1 was transfected with CRISPR constructs similar to , see material and methods for details. Transfection with a guide RNA in the third exon of CTNNB1 resulted in a population with reduced SGFP2 fluorescence and a population with increased fluorescence (B) compared to a Cas9 only control (A). Co-transfection of a single stranded oligo repair, led to a reduction in the population with decreased SGFP2 fluorescence (representing cells repaired with NHEJ that resulted in a loss of the SGFP2-CTNNB1 fusion protein) and an increase in the population with increased SGFP2 fluorescence (representing cells repaired with NHEJ that resulted in a stabilizing SGPF2-CTNNB1 truncation, mutation, insertion or deletion as well as cells with the correct HDR resulting in the stabilizing S45F mutation). Cells with increased fluorescence were sorted as single haploid cells (see ) and expanded for further analysis D) Graphical representation of part of the 3 rd exon of SGFP2-CTNNB1 before (top) and after (bottom) CRISPR/Cas9-mediated mutagenesis. Sanger sequencing is shown for one correctly targeted clone. The mutation changes the 45 th serine to a phenylalanine (S45F) and additionally removes the gRNA binding site and a silent mutation that introduces a HpaII restriction site for screening purposes. Mutated bases are indicated in light red. 14/32 single cell clones showed the expected genotyping fragment size and 7/32 showed large insertions or deletions. 7/14 clones showed the expected genotyping band contained the HpaII restriction site, 3/14 did not. Sanger sequencing coverage for the exon3 locus was obtained for 6/7 clones and all showed correct targeting (as shown for 1 clone here). Five clones were further analyzed; Clone 2,3,16 and 24 are from one single transfection, clone 27 from an independent transfection. E) Western blot, showing SGFP2-CTNNB1 WT (WT) and SGFP2 CTNNB1 S45F (S45F clone) protein levels. All panels are from one blot that was cut at the 70 kDa mark. Top: HAP1 SGFP2-CTNNB1(WT) and HAP1 SGFP2-CTNNB1(S45F) clones express the SGFP2-CTNNB1 protein at the expected height. Bottom: alpha-Tubulin (TUBA) loading control. A representative image of n=3 independent experiments is shown. F) Quantification of Western blots from n=3 independent experiments, including the one in, normalized to the SGFP2-CTNNB1 WT control (E), showing that the S45F clones express higher levels of SGFP2-CTNNB1 than their HAP1 SGFP2-CTNNB1(WT) parental line, even after 24h treatment with 8µM CHIR99021. Horizontal bar indicates the mean. G) Graph depicting the results from a MegaTopflash dual luciferase reporter assay, showing increased levels of TCF/LEF reporter gene activation for and HAP1 SGFP2-CTNNB1(S45F) , comparable to HAP1 SGFP2-CTNNB1(WT) cells treated for 24 hours with 8µM CHIR99021 treatment. Data points from n=3 independent experiments are shown. Horizontal bar indicates the mean. Values are depicted relative to the DMSO control, which was set to 1 for each individual cell line. H)

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Mutagenesis, Selection, Transfection, CRISPR, Construct, Fluorescence, Cotransfection, Sequencing, Binding Assay, Clone Assay, Western Blot, Luciferase, Reporter Assay, Activation Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Cytoplasmic complex characteristics in absence of SGFP2-CTNNB1 N-terminal phosphorylation. The S45F mutant was introduced using CRISPR (see ) and CHIR treated and control cells were measured after 24 hours. Details on sample size and statistics can be found in supplementary file 1. A) Graph depicting the fraction of particles with the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS for S45F mutant B) Graph depicting the speed of the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS for S45F mutant. C).Graph depicting the fraction of particles with the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS after 24h treatment with CHIR99021 C) Graphs depicting the speed of the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS after 24h treatment with CHIR99021. E-F) Graphs depicting the molecular brightness of SGFP2-CTNNB1 in the cytoplasm and nucleus relative to controls as measured with N&B in the same subcellular compartments for S45F mutant CTNNB1 (E) or after 24 hours of CHIR99021 treatment (F). EGFP monomer was used for normalization and EGFP dimer and trimer as controls for N&B measurements. The following supplements are available for : : Generation and characterization of a S45F mutant cell line (HAP1 SGFP2-CTNNB1(S45F) )

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Mutagenesis, CRISPR, Diffusion-based Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: In silico and experimental perturbation of WNT signaling. Details on experimental sample size and statistics can be found in supplementary file 1. A) Representative confocal images of HAP1-SGFP2WT (WT, top) and HAP1-SGFP2S45F (S45F, bottom) cells acquired with the same image settings. The S45F mutation leads to the accumulation and nuclear enrichment of CTNNB1 in the cell. B) Graph depicting the total concentration of SGFP2-CTNNB1 particles (monomeric plus complexed) as measured with FCS. C-D) Inhibition of CTNNB1 degradation is modelled as a reduction in the value of k3. C) Graph depicting the predicted total cytoplasmic CTNNB1 concentration as a function of k3. A reduction in k3 from 0.0068 ( , WNT ON and WNT OFF conditions) to ∼0.0043 (dotted line) corresponds to the cytoplasmic concentration observed (solid line). D) Graph depicting the predicted total nuclear CTNNB1 concentration as a function of k3. The solid horizontal line indicates the concentration measured for the S45F mutant by FCS. Note that the value of k3 that matches the observed cytoplasm concentration (dotted line) does not match the experimentally determined concentration in the nucleus (solid line). E-F) Graphs depicting the fraction of particles with the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) measured by FCS for wild-type and S45F mutant (E) and after 24-hour CHIR99021 treatment (F). The increase in the bound fraction in the oncogenic mutant or after GSK3 inhibition we find, is comparable to what we observed in WNT3A stimulated cells . G) Graph showing the predicted nuclear bound fraction of CTNNB1as a function of k3 with the TCF/CTNNB1 binding affinity of the model for WNT OFF (k9/k8 =320, pink line) and for WNT ON (k9/k8=33.5, blue line). Note that for WNT ON, the value for the nuclear bound fraction approximates the experimentally determined slow fraction for the S45F mutant (solid line, panel E) at the value for k3 that matches the cytoplasmic concentration of CTNNB1 (dotted line). H) Graph showing the predicted nuclear/cytoplasmic (N/C)-ratio as a function of k3 with TCF/CTNNB1 binding affinity of the model for WNT OFF (k9/k8=320, pink line) and WNT ON (k9/k8=33.5, blue line). Note that, although for WNT ON the value of the N/C-ratio increases with k3, there is still nuclear exclusion (N/C-ratio lower than 1, dashed line) at the value of k3 that matches the cytoplasmic CTNNB1-concentration (dotted line). I-K) The N/C-ratio as measured by FCS for wild-type and S45F mutant (I), after 24-hour CHIR99021 treatment (J) and after 4 hour WNT3A treatment (K). Note that all perturbations lead to nuclear accumulation (N/C-value exceeding 1). L) Graph showing the predicted N/C-ratio as a function of k3 with the WNT ON value for k9/k8 with the nuclear shuttling ratio of the model (k6/k7 ),corresponding to WNT OFF (k6/k7=0.96, pink line) and WNT ON (k6/k7=1.17, blue line), respectively. Note that the WNT ON value of k6/k7 increases the N/C-ratio to nuclear accumulation at the value for k3 that matches the cytoplasmic concentration (dotted line). M) Graph depicting the predicted total cytoplasmic CTNNB1 concentration as a function of k3 with WNT ON and WNT OFF values for k9/k8 and k6/k7Note that modulation of k9/k8 and k6/k7 has no effect on the predicted cytoplasmic concentration of CTNNB1. The horizontal solid line is the experimentally determined cytoplasmic CTBNN1 concentration (cf. panel B); the vertical dotted line is at the value of k3 that best reproduces this experimental finding in the model. N) Graph depicting the predicted total nuclear CTNNB1 concentration as a function of k3 for WNT ON and WNT OFF values for k9/k8 and k6/k7. Note that if both k9/k8 and k6/7 are changed from their WNT OFF values the predicted nuclear concentration of CTBNN1 better matches the experimentally determined concentration (horizontal solid line) at the value for k3 that matches the cytoplasm concentration (vertical dotted line).

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: In Silico, Mutagenesis, Concentration Assay, Inhibition, Diffusion-based Assay, Binding Assay

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: Live imaging of HAP1 SGFP2-CTNNB1 upon CHIR99021 stimulation. A) Representative stills from confocal time-lapse experiments corresponding to Supplementary Movies 3, showing an increase of SGFP2-CTNNB1 after treatment with 8µM CHIR99021. Scale bar = 20 μm. B-D) Quantification of time-lapse microscopy series, using the segmentation pipeline shown in . Arrow indicates the moment of starting the different treatments (T, see legend in B for details). B-C) Graph depicting the normalized intensity of SGFP2-CTNNB1 in the cytoplasm (B) or nucleus (C) over time. Solid lines represent the mean normalized fluorescence intensity and shading indicates the 95% confidence interval. n=166-400 cells for each condition and time point, pooled data from n=3 independent biological experiments. D) Graph depicting the nuclear/cytoplasmic ratio of SGFP2-CTNNB1 over time, calculated from raw intensity values underlying (B) and (C). This experiment was recorded simulateously with the data shown in . Additional data representation of this experiment is found in and Supplementary movies 4-6.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Imaging, Time-lapse Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

doi: 10.1101/2020.05.28.120543

Figure Lengend Snippet: additional biophysical properties of SGFP2-CTNNB1 S45F and SGFP2-CTNNB1 under CHIR99021 stimulation. The S45F mutant was introduced using CRISPR (see ) and CHIR treated and control cells were measured after 24 hours. Details on sample size and statistics can be found in supplementary file 1. A-B) Graph depicting the total number of SGFP2-CTNNB1 particles as measured with N&B upon S45F mutation (A) or CHIR99021 stimulation (B). C) Graph depicting the total concentration of SGFP2-CTNNB1 particles (monomeric plus complexed) as measured with FCS upon CHIR99021 treatment. D-E) Graphs depicting the speed of the second diffusion component (i.e. SGFP2-CTNNB1 containing complex) in the nucleus measured by FCS for S45F mutant (D) or 24 hour CHIR99021 treatment (E). E-F) Graph depicting the molecular brightness of SGFP2-CTNNB1 in the nucleus relative to controls as measured with N&B. EGFP monomer was used for normalization and EGFP dimer and trimer as controls for N&B measurements.

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433)).

Techniques: Mutagenesis, CRISPR, Concentration Assay, Diffusion-based Assay