Fig 2: YY1 directly transactivated KTN1 via binding to its promoter n BCa. (A) Using the UCSC and JASPAR databases analysis of predicted binding sites, including wild‐type (WT) and mutant (Mut) versions of these binding motifs. (B) Electrophoretic mobility shift (EMSA) assay analysis of the direct binding between the purified YY1 protein and the KTN1 promoter. The black arrow represents the binding complex between YY1 and a probe, and the white arrow represents the supershift generated by the association of the anti‐YY1 antibody with YY1 and the probe. (C) ChIP assay analysis of YY1 enrichment on the promoter of KTN1 in MDA‐MB‐231 cells compared with that using the immunoglobulin G mouse antibody. The different regions containing different putative YY1‐binding sites from left to right are shown. The binding enrichment of YY1 at the above binding sites on the promoter region was detected after knockdown of YY1. Error bars are shown with the s.d., n ≥ 3. * P < 0.05, ** P < 0.01, and *** P < 0.001. A two‐tailed t‐test or ANOVA was used to assess the P‐values

Fig 3: Overexpressed YY1 induced tumor aggressive growth in breast cancer (BCa). (A) Overexpression of YY1 promoted MDA‐MB‐231 cell growth in mice xenograft models compared with that in the vector only group. (B) Tumor volumes were measured after injected MDA‐MB‐231 cells treated with the YY1 overexpression plasmid in the xenograft mouse model (n = 3). (C) qRT‐PCR analysis of the expression levels of YY1 mRNA in the xenograft tumors. (D) Western blotting analysis of the levels of YY1, KTN1, and epithelial‐to‐mesenchymal transition (EMT) markers. (E) Hematoxylin and eosin (HE) staining analysis of the effects on tumor cell aggression of YY1 overexpression in vivo; a black arrow represents the range of infiltrating of tumor cells. (F) Immunohistochemistry (IHC) staining of YY1, KTN1, and EMT marker proteins in xenograft tumors. (G) A model of the YY1/DDX3X/KTN1‐regulatory axis in BCa development. Error bars are shown with the s.d., n ≥ 3. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the negative control group. A two‐tailed t‐test or ANOVA was used to assess the P‐values. Scale bars, 100 μm

Fig 4: High YY1 expression was associated with poor clinical prognosis. (A) Immunohistochemistry (IHC) staining of YY1 in breast cancer (BCa) tissues. All data are shown as the means of the 95% confidence interval plus the s.d. (B) The expression level of YY1 mRNA was examined by qRT‐PCR in 36‐paired BCa tissues compared to adjacent normal tissues. (C) Western blotting analysis of the level YY1 protein in MCF10A, MCF‐7, T47D, ZR75‐1, MDA‐MB‐453, HCC38, BT20, MDA‐MB‐231, and BT549 cell lines. (D) Kaplan–Meier analysis of well differentiated, moderately differentiated, and poorly differentiated for relapse‐free survival (RFS) with high versus low expression levels of YY1 mRNA from the GEPIA database. (E) Correlation analysis between YY1 and KTN1 from the tissue microarray (R = 0.896, P = 0). (F) Knockdown of YY1 in MDA‐MB‐231 and BT549 cell lines treated with negative control (siNC), YY1_1 and YY1_2 siRNA oligonucleotides was determined using western blotting assays. (G) Overexpression of YY1 in both BCa cell lines transfected with empty vector or YY1 overexpression plasmid was detected using western blotting analysis. The P‐value of data showed significant differences as indicated using * P < 0.05, ** P < 0.01 and *** P < 0.001

Fig 5: Depletion of YY1 blocked the invasive growth of breast cancer (BCa) cells in vitro and in vivo. (A) Knockdown of YY1 in MDA‐MB‐231 cells treated with siYY1_1 or siYY1_2 oligonucleotides compared with the siNC group as assessed by a qRT‐PCR assay. (B) CCK‐8 assay showing the proliferation of MDA‐MB‐231 cells treated with siNC and siYY1 oligonucleotides. (C) Monolayer colony formation assay showing the colony forming efficiencies of MDA‐MB‐231 cells treated with siNC and siYY1 oligonucleotides. (D) Transwell assay showing the migration and invasion of MDA‐MB‐231 cells treated with siNC and siYY1 oligonucleotides. (E) Western blotting analysis of protein levels in YY1‐overexpressing MDA‐MB‐231 cells treated with siKTN1 oligonucleotides. (F) Migration and invasion analysis in ‐overexpressing MDA‐MB‐231 cells treated with siKTN1 oligonucleotides using a Transwell assay. (G) Knockdown of YY1 with siYY1 oligonucleotides attenuated MDA‐MB‐231 cell growth in a mouse xenograft model compared with that in the siNC group. (H) Tumor volumes were measured after injection of MDA‐MB‐231 cells with siYY1 oligonucleotides in the xenograft mouse model; n = 6. (I) The expression levels of YY1 mRNA in the xenograft tumors. (J) Western blotting assay to detect the protein levels of YY1, KTN1, and epithelial‐to‐mesenchymal transition (EMT) markers. (K) Immunohistochemistry (IHC) staining of YY1, KTN1, and EMT marker in xenograft tumors. Error bars are shown with the s.d., n ≥ 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the negative control groups. A two‐tailed t‐test or ANOVA was used to assess the P‐values. Scale bars, 100 μm