Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells

(B) Cell lysates of indicated HCT116 cell lines analyzed by Western blotting with a β-catenin antibody; β-actin served as a loading control. (C) HCT116 β-catenin^{WTClover/∆45Cherry} (clone #37) immunoprecipitation with GFP, Cherry and control beads, or with a β-catenin antibody followed by immunoblotting with indicated antibodies. Representative results from three independent experiments are shown. Figure 2—figure supplement 1: Validation of endogenously fluorescent-tagged β-catenin in HCT116 colon cancer cells. (A) HCT116 β-catenin^{WTClover/∆45Cherry} (clone #37) cells express comparable amounts of β-catenin to the parental HCT116 WT cells. (B–D) Immunoprecipitation using HCT116 β-catenin^{WTClover/∆45} (clone #33 – left), β-catenin^{WT/∆45Cherry} (clone #45 – middle), and β-catenin^{WTClover/∆45} (clone #24 – right) were performed with GFP/Cherry or control beads, followed by Western blotting with the indicated antibodies. Representative results from three independent experiments are shown.

(A) Proliferation of the indicated HCT116 cell clones was monitored by live-cell imaging using an Incucyte instrument. (B) In HCT116 β-catenin^{WTClover/∆45Cherry}, mRNA-levels of CTNNB1, AXIN2, Cherry, and Clover were determined by RT-qPCR in control conditions and upon depletion of CTNNB1/β-catenin by siRNA (n=5; mean ± SD). Figure 3—figure supplement 1: Validation of the physiological function of fluorescently tagged β-catenin. (A) In HCT116 β-catenin^{WTClover/∆45} and β-catenin^{WT/∆45Cherry} mRNA-levels of CTNNB1, AXIN2, CHERRY, and CLOVER were determined by RT-qPCR upon silencing of CTNNB1/β-catenin. Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45} and β-catenin^{WT/∆45Cherry} after siRNA-mediated knockdown of CTNNB1 is shown (n=3; mean ± SEM; scale bar: 100 μm).

(E) Immunoprecipitation of HCT116 clone β-catenin^{WTClover/∆45Cherry} with E-cadherin confirms its interaction with β-catenin. Representative results from three independent experiments are shown. Figure 3—figure supplement 1: Validation of the physiological function of fluorescently tagged β-catenin. (C) Immunoprecipitation of HCT116 β-catenin^{WTClover/∆45} and β-catenin^{WT/∆45Cherry} with E-cadherin validates its interaction with β-catenin. A representative immunoblot is displayed.

(A) Left: Scheme showing the mode of action of GSK3β inhibitor CHIR99021 and CBP inhibitor ICG-001. Right: Indicated HCT116 cell lines were treated with 10 μM CHIR99021 and 10 μM ICG-001 for 24 hr, then Wnt activity was determined by a luciferase-based TCF4/Wnt-reporter assay (upper panel) and quantification of AXIN2 mRNA-levels by RT-qPCR (n=3; mean ± SD). (B) Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45Cherry} after 24 hr treatment with 10 μM CHIR99021 and 10 μM ICG-001 is shown (scale bar: 10 μm). The graph on the right depicts the ratio of nuclear to cytoplasmic fluorescent signal intensity for Clover and Cherry in HCT116 β-catenin^{WTClover/∆45Cherry}. Data from three independent experiments, each with at least 250 cells per condition, are shown as mean ± SEM. Every experiment includes at least 250 cells per condition. Enlarged representative images are shown in the Figure 4—figure supplement 2. (C) HCT116 β-catenin^{WTClover/∆45Cherry} were treated with LGK974 for 80 hr then 200 ng/ml of recombinant Wnt3a was added for 16 hr. Intensities of the Clover or Cherry signals were measured per slide and normalized to the intensity of Hoechst staining and to the control. 10–20 slides were measured per condition in one experiment. Data of three independent experiments are shown as mean ± SEM. Each dot represents an independent experiment. Scale bar: 10 μm. Figure 4—figure supplement 1: Tagging of β-catenin does not affect its functionality in canonical Wnt signaling. Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45} and β-catenin^{WT/∆45Cherry} after 24 hr treatment with 10 μM CHIR99021 and 10 μM ICG-001 is shown. The graphs on the right show the ratio of nuclear to cytoplasmic fluorescence intensities for Clover and Cherry in β-catenin^{WTClover/∆45} and β-catenin^{WT/∆45Cherry}, respectively (n=3 and 4; mean ± SEM). WRE, Wnt responsive element. Scale bar: 10 μm.

(A) Expression levels of AXIN2, CTNNB1/β-catenin, Cherry, and Clover were measured 72 hr after knockdown with siRNAs directed against Clover, Cherry, or both in HCT116 β-catenin^{WTClover/∆45Cherry} (n=4, mean ± SD). (B) Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45Cherry} upon transfection with siRNAs targeting CTNNB1, CLOVER, CHERRY, or a combination of CLOVER and CHERRY (scale bar: 25 μm). Intensities of the Clover or Cherry signals were measured per slide and normalized to the intensity of Hoechst staining. 5–10 slides were measured per condition and are shown as dots. Representative one from four independent experiments (Figure 5—figure supplement 1) is shown. Figure 5—figure supplement 1: Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45Cherry} upon transfection with siRNAs targeting CTNNB1, CLOVER, CHERRY, or a combination of CLOVER and CHERRY. Representative images from Figure 5. Scale bar: 25 μm. Immunofluorescence analysis of HCT116 β-catenin^{WTClover/∆45Cherry} upon transfection with siRNAs targeting CTNNB1, CLOVER, CHERRY, or a combination of CLOVER and CHERRY (scale bar: 25 μm). Intensity of Clover or Cherry signal was measured per slide and normalized to the intensity of Hoechst staining and then normalized to control. 5–10 slides per condition were measured. Data of four independent experiments are shown as mean ± SEM. Each dot represents an independent experiment.

(A) Concentrations (left) and diffusion coefficients (right) of Clover-tagged wild-type β-catenin and Cherry-tagged mutant β-catenin in the cytosol of HCT116 β-catenin^{WTClover/∆45Cherry}. (C) Concentrations (left) and diffusion coefficients (right) of Clover-tagged wild-type β-catenin and Cherry-tagged mutant β-catenin measured on HCT116 β-catenin^{WTClover/∆45Cherry} cells that were treated for 14–26 hr with either 10 µM CHIR99021 or DMSO as control. (D) HCT116 β-catenin^{wtClover/∆45Cherry} cells were treated with 5 µM of LGK974 for 96 hr. Sixteen hours before termination of the experiment, 200 ng/ml of recombinant Wnt3a was added. FCS analysis was performed in the cytosol (data shown here) and in the nucleus (data shown in Figure 6—figure supplement 1). Each data point represents a 120 s FCS measurement in a single cell. In total, more than 40 cells per condition were measured in three independent experiments per box plot. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). See also Supplementary file 1. Figure 6—figure supplement 1: Cross-correlation analysis reveals that the two β-catenin protein isoforms diffuse independently. (A) Shown are representative autocorrelation curves (green, red) and the cross-correlation (blue) from 120 s FCS measurements in the cytosol and the nucleus of HCT116 β-catenin^{WTClover/∆45Cherry}. The non-zero amplitudes of the autocorrelation curves in the green and red color channels indicate the presence of both β-catenin isoforms in the cytosol and in the nucleus. Jagged lines: experimental data, smooth lines, fits with a model function, y-axis: amplitudes of the pair correlation functions. (B) HCT116 β-catenin^{WTClover/∆45Cherry} cells were treated with 5 µM of LGK974 for 96 hr. Sixteen hours before termination of the experiment, 200 ng/ml of recombinant Wnt3a was added. Afterward, FCS measurements were performed in the cytosol (data shown in Figure 6) and in the nucleus (shown here). For each data point, a 120 s FCS measurement was carried out in a single cell. Per box plot, more than 40 cells were examined in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant) The exact values are provided as the Supplementary file 1. FCS, fluorescence correlation spectroscopy.

(B) HCT116 β-catenin^{WTClover/∆45Cherry} and sgAPC targeted clone (APC^LOF) cells were treated for ~16 hr with either 10 µM CHIR9901 or DMSO as control. Subsequently, FCS measurements were performed in the cytosol (data shown here) and in the nucleus (data shown in Figure 7—figure supplement 1A). Each data point in the box plots represents a result from a 120 s FCS measurement in a single cell. Per box plot, more than 40 cells were investigated in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). The exact values are provided in the Supplementary file 1. Figure 7—figure supplement 1: Truncation of APC affects abundance and diffusion of wild-type but not mutant β-catenin in the nucleus. (A) HCT116 β-catenin^{WTClover/∆45Cherry} and sgAPC targeted clone (APC^LOF) cells were treated for ~16 hr with either 10 µM CHIR9901 or DMSO as control. Afterward, FCS measurements were performed in the cytosol (data shown in Figure 7) and in the nucleus (shown here). For each data point, a 120 s FCS measurement was carried out in a single cell. Per box plot, more than 40 cells were examined in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). The exact values are provided as the Supplementary file 1. FCS, fluorescence correlation spectroscopy.

Figure 7—figure supplement 1: (B) Both β-catenin alleles of HCT116 cells bind to APC, GSK3β, and Axin1. IP with anti-APC antibody was performed GFP/Clover and Cherry were detected (left panel). IPs with RFP/Cherry and GFP/Clover beads were performed and β-catenin, Axin1, and GSK3β were detected (right panel). Representative experiments from three independent are shown.