Fig. 8

ACTN4 sustains CSCs properties by promoting β-catenin stabilization. (A) The expressions of ACTN4 and β-catenin pathway signaling were elevated in the sorted ALDEFLUOR-positive and CD44⁺/CD24⁻ cells; (B) ACTN4 knockdown in CSCs resulted in decreased β-catenin and p-AKT/GSK3β expressions; (C) a. the immunofluorescence assay showed the co-localization of ACTN4 and β-catenin was mainly located in the cytosol (arrowheads); b. the immunoprecipitation assay revealed the direct molecular interaction between β-catenin and ACTN4; (D) Breast CSCs were transfected with shACTN4, treated with CHX (10 μg/ml), and MG132 (10 μM) for the indicated time and immunoblotted, and the results showed that ACTN4 silencing could promote β-catenin proteasome degradation. A quantitative measurement was conducted to further analyze with Image J (**P < 0.01, values represented as the mean ± SD, n = 3). After CHX treatment, the half-life of β-catenin degradation in shCtrl cells was estimated to be approximately 12.1 h, whereas it was 4.5 h in ACTN4 silencing cells based on Engauge Digitizer software analysis; (E) ACTN4 overexpression caused a loss of poly-ubiquitination accumulation of β-catenin, while its knockdown was found to increase β-catenin ubiquitination and its consequent degradation, presenting as accumulation of ubiquitinated β-catenin; (F) Breast CSCs transfected with shACTN4 were treated with LY294002 (10 μM) and LiCl (10 μM) and immunoblotted, and the results indicated that ACTN4 silencing activated β-catenin proteasome degradation via modulating GSK3β/Akt phosphorylation