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. 2020 Jun 3;123(4):633–643. doi: 10.1038/s41416-020-0873-z

Fig. 5. ChIP-Seq and luciferase activity analyses of SLFN5 inhibition of ZEB1 transcription by binding to specific sites within the ZEB1 promoter region.

Fig. 5

a Schematic diagram of luciferase reporter gene constructs with the ZEB1 promoter (TSS upstream from –1 to –2000 bp) or the indicated length promoters. b, c Relative luciferase activity analyses of the ZEB1 promoter with the indicated length in MCF7 cells stably transfected with control shRNA or SLFN5 shRNA (b), or in MDA-MB-231 cells stably transfected with lentiviral control or lentivirus-overexpressing SLFN5 (c). **P < 0.01. d ChIP-Seq analysis of genetic features of chromosome fragments immunoprecipitated by SLFN5 antibody in MCF7 cells. e Putative SLFN5-binding motifs with the top-10 scores according to ChIP-Seq analysis. f ChIP-Seq data analysis of binding peaks of SLFN5 at promoters of ZEB1, ZEB2, SNAI1, SNAI2, TWIST1, TWIST2 and CDH1 (E-cadherin) in MCF7 cells with cell lysate input as a control. g Real-time PCR analyses of the ZEB1 promoter in SLFN5 antibody-immunoprecipitated chromosome DNA. Cell lysate input was used as a positive control, and IgG immunoprecipitation was used as a negative control. **P < 0.01. h Schematic diagram of luciferase reporter gene constructs with ZEB1 promoter mutants of the putative SLFN5-binding site #1. The black box indicates an intact wild-type binding site, and the red box indicates a mutated binding site. i, j Relative luciferase activity analyses of ZEB1 promoter mutants of the putative SLFN5-binding site #1 in MCF7 cells stably transfected with control shRNA or SLFN5 shRNA (i), or in MDA-MB-231 cells stably transfected with lentiviral control, full-length wild-type SLFN5 expression vector or C-terminal deletion mutant (SLFN5delC) expression vector (j). **P < 0.01. n.s. indicates non-significant.