Journal: bioRxiv

Article Title: CD73 restrains mutant β-catenin oncogenic activity in endometrial carcinomas

doi: 10.1101/2024.11.18.624183

Figure Lengend Snippet: ( A-C ) HEC-1-A cells were transfected with Xenopus β-catenin ΔEX3 and NT (non-targeting) or NT5E siRNA and cultured in 1% O 2 and 5% CO 2 for 48 hours. (A) Representative immunofluorescence images used in quantifying nuclear localization of myc-β-catenin ΔEX3 . Scale bar: 50 µm. Fluorescence intensity was determined with BZ-X800 Analyzer Macro cell count software (Keyence). ( B) Validation of CD73 knockdown and ( C ) nuclear fluorescence intensity of myc-β-catenin ΔEX3 . ( D-F ) Representative immunoblots of n = 2 independent experiments of cellular fractionations from NT5E WT and NT5E KO HEC-1-A cells. Cells were transfected with ( D ) Xenopus β-catenin ΔEX3 or patient-specific β-catenin mutants (E) S37F or (F) G34R. Graphs show densitometry for myc-β-catenin mutant expression for each cellular fraction normalized to myc-β-catenin mutant expression in the whole cell lysate (WCL). Cellular fraction markers: Rab11a (membrane), SP1 (nuclear), and H2AX (chromatin). ( G ) mRNA and ( H ) protein expression of differentially expressed cell-cell adhesion components in NT5E WT and NT5E KO HEC-1-A cells. Data represent the mean ± SEM. ****P < 0.0001, Mann-Whitney test; **P < 0.01, ***P < 0.005; Welch t-test.

Article Snippet: Relative intensity for each image was measured using Keyence macro cell counting software.

Techniques: Transfection, Cell Culture, Immunofluorescence, Fluorescence, Cell Counting, Software, Knockdown, Western Blot, Mutagenesis, Expressing, Membrane, MANN-WHITNEY