wild type flag axin1  (Addgene inc)

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Deletion of the GSK3β and β-catenin–binding domains strongly increases β-catenin signaling. A, Schematic diagram depicting the FLAG-tagged AXIN1 mutant expression vectors that were generated. Amino acid numbers are based on the long 862 amino acid isoform of AXIN1. For clarity, we refer to each domain by one reported binding partner, but especially for the MEKK1, CK1, and PP2A domains; additional binding proteins have been reported. B, Immunoblot for FLAG-tagged AXIN1 to demonstrate expression of the expected variants after HEK293T transfection. Tubulin was used as loading control. C, A β-catenin reporter assay was conducted in HEK293T cells expressing WT AXIN1 and variants lacking the depicted domains. The β-catenin reporter activities are shown as WRE/CMV- Renilla ratios (in triplicate, three independent experiments), in which the value obtained for wild-type AXIN1 was arbitrarily set to 1. As a positive control for increased β-catenin signaling, Wnt3A conditioned medium was added to empty vector (EV)-transfected cells (EV+Wnt3A). Data are shown as mean ± SD. Statistical significance was analyzed using a Mann–Whitney test. ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Binding Assay, Mutagenesis, Expressing, Generated, Western Blot, Transfection, Reporter Assay, Positive Control, Plasmid Preparation, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Analysis of β-catenin signaling in cell clones with endogenous knockout of the GSK3β and β-catenin domains. HEK293T and SNU449 AXIN1-repaired cells were used to knockout the GSK3β-binding domain encoded by exon 5. We also identified one HEK293T clone with an endogenous knockout of the β-catenin–binding region. A, Immunoblot using an N-terminal AXIN1 antibody (cat. #3323, Cell Signaling Technology) to show expression of the shortened AXIN1 proteins. β-Actin served as a loading control. B, qRT-PCR assay to demonstrate the mRNA expression level of AXIN2 (in triplicate, n = 2 independent experiments). Expression levels were depicted relative to the housekeeping gene GAPDH . The value for the WT control was arbitrarily set to 1. C, A β-catenin luciferase reporter assay was performed to determine the β-catenin signaling activity in all knockout clones. Values are depicted as WRE/MRE ratios (in triplicate, n = 3 independent experiments). The value for the WT control was arbitrarily set to 1. D, siRNA-mediated knockdown of AXIN2 was performed in all knockout clones, followed by a β-catenin reporter assay. Values are depicted relative to the WRE/CMV- Renilla ratios obtained for the siControl-WT (five replicates, two independent experiments), which was arbitrarily set to 1. Data are shown as mean ± SD. Statistical significance for all experiments was analyzed using a Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Clone Assay, Knock-Out, Binding Assay, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Analysis of missense variants in the GSK3β-binding domain. Tumor-associated missense variants within the GSK3β-binding domain were tested for their effect on β-catenin signaling regulation. A, Schematic diagram indicating the sequential amino acid deletions within the GSK3β domain that were generated in the AXIN1 expression plasmid. B, A β-catenin reporter assay was performed to show the defect in β-catenin regulation associated with the variants shown in A . C, Immunoprecipitation experiment following cotransfection with HA-tagged GSK3β to identify which deletion variants shown in A affect GSK3β binding. AXIN1 and GSK3β were detected using anti-FLAG and anti-GSK3β antibodies, respectively. Transfection with empty vector (EV) and nontransfected cells were used as negative controls. D, Schematic diagram depicting the selected tumor-associated missense variants within the V383-T402 domain. These 12 variants were the only ones reported in the cBioPortal and COSMIC databases until January 2021. E, A β-catenin reporter assay to determine the defect in β-catenin regulation for the missense variants is shown in D . F, Immunoprecipitation experiment following cotransfection with HA-tagged GSK3β to identify which missense variants shown in D affect GSK3β binding. Image is a composite of two original blots (see Supplementary Data S1) in which the AXIN1 variants have been arranged in numerical order. G, Cartoon representation of GSK3β (gray) with bound Axin1 α-helix (deep teal). Right, details of the interaction interface with amino acid sidechains in stick representation with nitrogen atoms (blue) and oxygen (red). Dashed line, hydrogen bond between Aspartate D264 in GSK3β and Arginine R395 in Axin1. AXIN1 residues mutated in this study are labeled. All β-catenin reporter activities are depicted as WRE/CMV- Renilla ratios (in triplicate, two independent experiments), in which the value obtained for the empty vector was arbitrarily set to 1. Data are shown as mean ± SD. Statistical significance for all experiments was analyzed using a Mann–Whitney test. **, P < 0.01; ***, P < 0.001.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Binding Assay, Generated, Expressing, Plasmid Preparation, Reporter Assay, Immunoprecipitation, Cotransfection, Transfection, Labeling, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Missense variants in the β-catenin–binding domain mostly retain functionality. Tumor-associated missense variants within the β-catenin–binding domain were tested for their effect on β-catenin signaling regulation. A, Schematic diagram of the indicated sequential amino acid deletions that were generated in the AXIN1 expression plasmid. B, A β-catenin reporter assay was performed to show the defect in β-catenin regulation associated with the variants shown in A . C, Schematic diagram depicting the 15 selected tumor-associated missense variants within the R450_P503 domain. D, A β-catenin reporter assay to determine the defect in β-catenin regulation for the missense variants shown in C . Reporter activities are depicted as WRE/CMV- Renilla ratios (mean ± SD; three independent experiments), in which the value obtained for the empty vector (EV) was arbitrarily set to 1. E, Immunoprecipitation experiment with FLAG-tagged AXIN1 variants to identify which domain deletions affect endogenous β-catenin binding. F, Immunoprecipitation experiment with depicted FLAG-tagged AXIN1 missense variants to determine β-catenin–binding capacity. Deletion of the N-terminal half of AXIN1 up to the β-catenin domain (M1_G430del) was used to allow a better evaluation. A larger deletion (M1_P503del) also removing the β-catenin domain was used as negative control. Cotransfected GFP–β-catenin and endogenous β-catenin were detected using a β-catenin antibody. In all IP experiments, transfection with EV and nontransfected cells was used as negative controls. Data are shown as mean ± SD. G, Cartoon representation of β-catenin (gray) with bound AXIN1 α-helix (deep teal). Side chains of amino acid residues that are mutated in this study are indicated in stick representation, with nitrogen atoms (blue) and oxygen (red). Right, close-up of the region around AXIN1 residue Valine 478. Statistical significance for all experiments was analyzed using a Mann–Whitney test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Binding Assay, Generated, Expressing, Plasmid Preparation, Reporter Assay, Immunoprecipitation, Negative Control, Transfection, Residue, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Analysis of missense variants within the RGS/APC domain. Tumor-associated missense variants in the RGS/APC domain were analyzed for their defect in β-catenin regulation, APC and β-catenin binding, and capacity to induce intracellular puncta formation. A, A β-catenin reporter assay was performed to determine the defect in β-catenin regulation for 37 RGS/APC domain variants. Red-marked bars represent variants that show a near-complete loss of GFP-APC and/or β-catenin binding (see ), whereas orange bars represent variants with partial loss. All β-catenin reporter activities are depicted as WRE/CMV- Renilla ratios (in triplicate, three independent experiments), in which the value obtained for the empty vector (EV) was arbitrarily set to 1. B, Immunoprecipitation assay to determine the binding capacity of missense variants to cotransfected GFP-APC or endogenous β-catenin. C, Immunofluorescence analysis of transfected AXIN1 missense variants to determine their puncta formation capacity. Average dot size was determined using ImageJ software on at least 6 independent cells. Data are shown as mean ± SD. D, Surface representation of AXIN1 (gray) with bound APC helix (deep teal). Amino acid residues mutated in this study that are located on the surface of AXIN1 are labeled and colored according to extent of loss of APC binding , with yellow indicating no loss, orange indicating partial loss, and red indicating near-complete loss of APC binding. Statistical significance for all experiments was analyzed using a Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Binding Assay, Reporter Assay, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Transfection, Software, Labeling, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Immunohistochemical analysis of induced liver tumors in mice. An example is shown of two adjacent AXIN1–R395P-induced liver lesions. IHC was performed to reveal expression of transfected Myc-tagged AXIN1, endogenous location of β-catenin, and Ki67 to identify proliferating cells. Hematoxylin and eosin (H&E) staining revealed mostly slight increases in hepatocyte size and mild-to-moderate nuclear atypia. Scale bar, 250 μm.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Immunohistochemical staining, Expressing, Transfection, Staining

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Characterization of AXIN1-truncating mutations in regulating β-catenin signaling. Analysis of clones carrying various endogenous AXIN1 truncations generated in HEK293T cells using CRISPR/Cas9-mediated gene editing. A, Schematic diagram showing the AXIN1 truncation variants generated in HEK293T cells using gene editing. The red domain at the end of truncated variants represents amino acid stretches resulting from the introduced frameshift mutation. B, Immunoblot analysis of generated clones using AXIN1 antibodies with N- and more C-terminal epitopes (cat. # 3323S and cat. #2087S, Cell Signaling Technology). Images were generated from one blot for each antibody, with irrelevant lanes removed. Original blots can be seen in Supplementary Data S1. C, qRT-PCR assay to determine AXIN1 mRNA expression levels (in triplicate, two independent experiments). Expression levels are depicted relative to the housekeeping gene GAPDH . The value for the WT control was arbitrarily set to 1. D, A β-catenin reporter assay was performed to determine β-catenin signaling levels in AXIN1-truncated clones. Values are depicted as WRE/MRE ratios (mean ± SD; in triplicate; three independent experiments). The value for the WT control was arbitrarily set to 1. E, Following siRNA-mediated knockdown of AXIN2 , a β-catenin reporter assay was performed in all AXIN1-truncated clones. Values are depicted relative to the WRE/CMV- Renilla ratios obtained for the siControl-WT, which was arbitrarily set to 1. Note the logarithmic scale. Data are shown as mean ± SD. Statistical significance for all experiments was analyzed using a Mann–Whitney test. *, P < 0.05; **, P < 0.01; ns, nonsignificant.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Clone Assay, Generated, CRISPR, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing, Reporter Assay, MANN-WHITNEY

Journal: Cancer Research

Article Title: Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling

doi: 10.1158/0008-5472.CAN-23-2268

Figure Lengend Snippet: Co-occurrence of mutations in β-catenin–related genes observed in AXIN1-mutant HCC and colorectal cancer tumors. From the cBioPortal database, we obtained information from AXIN1-mutant HCC and colorectal cancer (CRC) tumors about APC , CTNNB1 , and BRAF mutation status, in addition to colorectal cancer tumor location and MSI-status. A, Identification of oncogenic β-catenin mutations in HCC tumors carrying either truncating ( n = 49) or missense ( n = 15) AXIN1 mutations. B, Identification of oncogenic β-catenin or inactivating APC mutations in colorectal cancer tumors carrying either truncating ( n = 15) or missense ( n = 58) AXIN1 mutations. The BRAF mutation (light blue) is the classical BRAF-V600E variant. Gray, microsatellite instability-high colorectal cancer tumors carrying a mismatch repair defect. Colorectal cancer tumor location is defined as being located on left- or right-sided of the splenic flexure. Identity of the specific β-catenin and APC mutations can be found in Supplementary Table S7.

Article Snippet: Wild-type (WT) FLAG-AXIN1 (cat. #109370, RRID:Addgene_109370) and HA GSK3β wt pcDNA3 (cat. #14753, RRID:Addgene_14753) were purchased from Addgene.

Techniques: Mutagenesis, Variant Assay