Journal: Molecular medicine reports

Article Title: miR‑200b‑3p inhibits proliferation and induces apoptosis in colorectal cancer by targeting Wnt1.

doi: 10.3892/mmr.2018.9287

Figure Lengend Snippet: Figure 6. Effect of miR‑200b‑3p on the activity of Ki67, β‑catenin and caspase‑3. (A) Relative caspase‑3 activity. (B) qPCR for the expression of Ki67 and β‑catenin. (C) Western blot analysis of the expression of pro caspase‑3 and cleaved caspase‑3. (D and E) Western blot analysis of the expression of Ki67 and β‑catenin. β‑actin was used as a loading control. *P<0.05, **P<0.01, ***P<0.001 vs. mock group. siRNA‑NC, small interfering RNA negative control; si‑Wnt1, small interfering RNA‑Wnt1; miR, microRNA.

Article Snippet: The membranes were incubated with primary antibodies overnight at 4 ̊C, and probed with secondary antibodies at room temperature for 1.5 h. The antibodies were as follows: Wnt1 (cat. no. sc-514531; 1:500; Santa Cruz Biotechnology, Inc.); β-catenin (cat. no. 2698; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA); Ki67 (cat. no. ab16667; 1:1,000 dilution; Abcam, Cambridge, UK); β-actin (cat. no. 3700; 1:1,000; Cell Signaling Technology, Inc.;); horseradish peroxidase‐conjugated secondary antibody (cat. no. sc-2954; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Expressing, Western Blot, Control, Small Interfering RNA, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Identification of Two Distinct Stem Cell Clusters, Lrig1-Derived and Wnt/CD44-Dependent, in Corneal Epithelium

doi: 10.3390/ijms26136383

Figure Lengend Snippet: ( A ) Beta-catenin (green fluorescence) and non-phospho beta-catenin (red fluorescence) staining in the cornea of WT versus CD44KO mice. Wnt pathway seems to be downregulated in the absence of CD44. ( B ) Lrig1 (green fluorescence) is still expressed in CD44KO mouse cornea (upper panel). ( C ) Cell lineage tracing experiments in WT versus CD44KO mice. Red = Tomato was activated in Lrig1+ cells upon tamoxifen treatment (lower panel). Note that 100% of cells are red (Lrig1-derived cells) in WT animals already after 3 days, however in CD44KO animals only 70% of cells are red (Lrig1-derived cells) after 3 days, and 100% red after 7 days (Lrig1-derived compartment is in blue). No difference was observed after 14 days. WT and CD44KO transgenic mice expressing an inducible form of the Cre recombinase under the control of the Lrig1 promoter and a Cre reporter transgene coding for the tomato fluorescent protein were injected twice with 2 mg of tamoxifen in the first 24 h in order to induce the recombinase activity and to remove the codon stop that prevents the constitutive expression of the tomato protein in Lrig1 expressing corneal epithelial cells. Animals were killed at each timepoint after the tamoxifen injections. Eyes were fixed overnight in formalin and then included in Tissue-Tek OCT compound, Sakura Finetek Europe Alphen aan den Rijn, The Netherlands, and stored frozen. Cornea sections were further prepared with a cryostat (−20 °C), dried, and mounted on histological slides with the DAPI fluoromount -G (c) (SouthernBiotech, Birmingham, AL, USA). Scale bars = 87 µm.

Article Snippet: The activation of Wnt signaling pathway, was investigated in WT versus CD44KO mice, by staining the normal beta-catenin (D10A8 XP, rabbit monoclonal antibody, Cell Signaling, Danvers, MA, USA) and the activated form (non-phosphorylated form) of beta-catenin (D13A1, rabbit monoclonal antibody, Cell Signaling, Danvers, MA, USA) in the cornea of WT and CD44KO mice.

Techniques: Fluorescence, Staining, Derivative Assay, Transgenic Assay, Expressing, Control, Injection, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Alix-mediated selective packaging of β-catenin into extracellular vesicles enhances their proangiogenic function

doi: 10.1016/j.jbc.2026.111254

Figure Lengend Snippet: Alix mediates the interaction with β-catenin . A , HeLa cells were transiently transfected with 3HA-Alix ( green ) and 3FLAG-β-catenin ( red ). After 48 h, the cells were fixed and stained with a rabbit anti-HA antibody, followed by incubation with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody and a mouse anti-FLAG antibody, followed by incubation with an isothiocyanate-conjugated goat anti-mouse secondary antibody. The bar represents 50 μm. B , HEK293T cells (4 × 10 6 ) were transfected with 3HA-Alix and 3FLAG-β-catenin, and coimmunoprecipitation was performed with the FLAG antibody after 48 h. Cell lysates and immunoprecipitates were analyzed via Western blotting with anti-FLAG and anti-HA antibodies. The full-length blots are presented in . Alix, apoptosis-linked gene 2–interacting protein X; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: Anti-HA (1:100 dilution, 51064-2-AP), anti-FLAG (1:100 dilution, 66008-4-Ig), and anti-β-catenin (1:100 dilution, 51067-2-AP) primary antibodies, along with their corresponding secondary antibodies, were all purchased from Proteintech.

Techniques: Transfection, Staining, Incubation, Western Blot