Fig 2: Knockdown of CTNND1 upregulates CXCR4 via activation of PI3K/AKT/HIF-1a pathway. (A) KEGG pathway analysis of RNA sequencing between cells with sh-NC and sh-CTNND1 in MDA-MB-231 and MDA-MB-468 cells. (B) Immunoblotting of verifying activation of PI3K/AKT/HIF-1a pathway. (C) Expression of CXCR4 by qRT-PCR (left) and immunoblotting (right) in cells with CTNND1 knockdown or BKM120. (D) Images and quantification of chemotaxis of cells with CTNND1 knockdown or BKM120 in the upper chamber and CXCL12 in the bottom chamber. Scale bar: 200 µm. (E) IHC staining of AKT, p-AKT and CXCR4 in the bone of mice withEO771CTNND1KD. Scale bar: 200 µm, T: tumor, B: bone, M: bone marrow. (F) Chromatin immunoprecipitation (ChIP) assay of the enrichment of HIF-1a at the CXCR4 promoter relative to IgG in MDA-MB-231 cells transduced with sh-CTNND1. (G) The enrichment of HIF-1a at the CXCR4 promoter relative to Input in MDA-MB-231 cells transduced with sh-CTNND1 by CHIP-qPCR. (H) Luciferase reporter assay for the CXCR4 promoter construct transfected in MDA-MB-231 cells. * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).

Fig 3: (A) The apoptosis of MDA-MB231, MDA-MB-468 with downregulation of CTNND1 analyzed by flow cytometry. (B) The colony formation of MDA-MB231, MDA-MB-468 with downregulation of CTNND1. (C) Expression of CTNND1 and CXCR4 in MDA-MB231 and MDA-MB-468 with CTNND1 reduction and PI3K inhibitor LY294002 by qRT-PCR. (D) Immunoblotting of the activation PI3K/AKT/HIF-1a pathway with downregulation of CTNND1 or LY294002 in MDA-MB231 and MDA-MB-468 cells. (E) The enrichment of ß-actin in MDA-MB-231 cells transduced with sh-NC or sh-CTNND1 by CHIP-qPCR. All experiments were repeated at least 3 times. *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).

Fig 4: (A) Cartoon exemplifying the silent mutations in the siRNA1 and siRNA2 target sequence of WT CTNND1 cDNA, which confer resistance to the siRNA1 and siRNA2 and no change in the amino-acid sequence of the resulting protein (mut-CTNND1), compared with the WT. (B) Images and quantification of chemotaxis of MDA-MB-231 and MDA-MB-468 cells expressing mut-CTNND1 despite co-transfection with siRNA1 and siRNA2. (C) Immunoblot of CTNND1 and CXCR4 in MDA-MB-231 and MDA-MB-468. Note the strong expression of CTNND1 in cells expressing mut-CTNND1 despite co-transfection with siRNA1 and siRNA2. (D) Expression of CTNND1 and CXCR4 by qRT-PCR in MDA-MB-231 and MDA-MB-468 transfected with the other two sh-CTNND1 plasmids. (E) Images and quantification of transwell migrated and invaded cells after CTNND1 knockdown by being transfected with the other two sh-CTNND1 plasmids. Scale bar represents 200 µm. All experiments were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).