Fig 2: Knockdown of ZBTB11 induces R-loop accumulation and DNA damage to suppress the growth of BC cells in a DDX1-dependent manner. (A) Rescue of DDX1 expression in ZBTB11 knockdown BC cells was achieved by transfecting a plasmid expressing Flag-tagged DDX1. The expressions of ZBTB11, Flag-tagged DDX1, and ?H2AX in DDX1-rescued BC cells were detected by Western blot analysis. (B) R-loop in ZBTB11 knockdown BC cells with DDX1 rescue was determined by Dot blot using S9.6 antibody. (C) R-loop and DNA damage in ZBTB11 knockdown BC cells with DDX1 rescue were determined by immunofluorescence, with antibodies against S9.6 and ?H2AX, respectively. Scale bar, 10 µm. (D, E) The proliferation and colony formation ability of DDX1-rescued ZBTB11 knockdown BC cells were evaluated by MTT assay and colony formation assay, respectively. The relative cell growth curves and numbers of colonies are shown. (F) Overexpression of RNase H1 in ZBTB11 knockdown BC cells was achieved by transfecting a plasmid expressing HA-tagged RNase H1. The expressions of ZBTB11 and HA-tagged RNase H1 in RNase H1-overexpressed BC cells were detected by Western blot analysis. (G, H) The proliferation and colony formation ability of RNase H1-overexpressed ZBTB11 knockdown BC cells were evaluated by MTT assay and colony formation assay, respectively. The relative cell growth curves and numbers of colonies are shown. Statistical significance was assessed using one-way or two-way analysis of variance.

Fig 3: In vivo tumorigenicity of DDX1-KO LoVo cells with or without exogenous DDX1. A, WT, DDX1-KO and DDX1-KO + DDX1 LoVo cells (5 × 106 cells) were subcutaneously injected into nude mice. The volume of each tumor was measured every week. B, Photographs of isolated tumors from the transplanted mice. C, Weight of isolated tumors from the transplanted mice. Each value represents the mean (n = 5) ± SD. ***P < .001. D, H&E and immunohistochemical staining data of paraffin-embedded sections of isolated tumors from the transplanted mice. Anti-PCNA antibody and DAPI were used for visualizing mitotic cells and nuclei, respectively. Scale bars = 50 µm

Fig 4: Interaction of DDX1 with the enhancer/promoter region of the LGR5 gene. A, Schematic diagram of reporter constructs. Genomic DNA fragments encompassing -2937 to +311 from the transcriptional initiation site of the human LGR5 gene were inserted into pGL3 vector. B, Luciferase activities of LoVo cell lysates were measured 48 h after transfection, and firefly vs Renilla (F/R) was calculated. C, WT LoVo cells were transfected with the indicated reporter constructs in combination with the DDX1 expression vector (+DDX1) or the empty vector (+Empty). Luciferase activities of cell lysates were measured 48 h after transfection, and firefly vs Renilla (F/R) was calculated. D, WT and DDX1-KO + DDX1 LoVo cells were subjected to ChIP assays using anti-DDX1 or control rabbit IgG. Relative abundance of the LGR5 gene-specific PCR product is shown. B-D, Each value represents the mean (n = 5) ± SD. **P < .01

Fig 5: 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.