FIGURE 4.

ZBTB11 transcriptionally regulates DDX1 in BC cells. (A) The differential genes from the RNA‐Seq data of ZBTB11 knockout T24 cells are shown in Venn diagrams. (B) The differential genes of ZBTB11 knockout T24 cells are shown as a volcano plot. ZBTB11 knockout significantly downregulated DDX1. (C) mRNA expression of different genes in ZBTB11 knockdown BC cells was evaluated by qRT‐PCR analysis. (D) Correlation between ZBTB11 and DDX1 in BC, as predicted by the GEPIA database (gepia.cancer‐pku.cn), is shown. (E) Protein expression of DDX1 in ZBTB11‐rescued ZBTB11 knockdown BC cells was evaluated by Western blot analysis. (F) The binding peaks of ZBTB11 in the promoter regions of DDX1 in K562 and MCF‐7 cells were visualized using the ENCODE database (www.encodeproject.org). (G) CGGAA‐containing sequences and mutated sequences in DDX1 promoter region. The DDX1 promoter sequence and the mutated sequences were individually cloned into the pGL3 vector for the luciferase reporter assay. (H) The luciferase activities of CGGAA‐containing sequences and of mutated sequences in ZBTB11 knockdown UMUC3 cells were assessed. Relative luciferase activities are presented as a bar chart. Statistical significance was assessed using one‐way analysis of variance. (I) UMUC3 cells were transfected with Flag‐ZBTB11△ZnF plasmids or wild‐type Flag‐ZBTB11 plasmids. Flag expression was detected by Western blot. ChIP qRT‐PCR analysis was performed to confirm whether ZBTB11 binds to the promoter region of DDX1 gene via the ZnF_C2HC domain.