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Fig. 6 | Molecular Cancer

Fig. 6

From: The pro-metastasis effect of circANKS1B in breast cancer

Fig. 6

ESRP1 promotes circANKS1B formation and it is also a target of USF1 in breast cancer. a qRT-PCR analysis of circANKS1B and linear ANKS1B in MCF-7 and MDA-MB-231 cells with ESRP1 overexpression. b Schematic of circANKS1B minigene with four wild-type (WT) or mutant (Mut) ESRP1 binding sites. c RIP analysis of ESRP1-binding to wild-type (WT) or mutant (Mut) circANKS1B minigene using an antibody against ESRP1. d qRT-PCR analysis of circANKS1B after co-transfection with si-ESRP1 or si-NC and wild-type (WT) or various mutant (Mut) circANKS1B minigenes. e Representative IHC images of low (patient # 56) and high (patient # 28) ESRP1 expression in breast cancer tissues. Scale bar = 20 μm. f A strong correlation between ESRP1 and circANKS1B expression in breast cancer tissues assessed by Spearman correlation coefficients (r = 0.626, n = 165, p < 0.001). g-h Schematic of insertion of ESRP1 binding sites (GGT-rich) and locations of primers used for segments of two genes (SYT8 and Snail) devoid of circRNAs (left). RT-PCR analysis of circSYT8, linear SYT8, circSnail and linear Snail in MDA-MB-231 cells transfected with SYT8 or Snail minigene, with or without si-ESRP1 or treatment with RNase R (middle). The formation of circSYT8 and circSnail through back-splicing was confirmed by Sanger sequencing, red arrow indicates the junction site (right). i Schematic of three putative USF1 binding sites in ESRP1 promoter region (above). ChIP-PCR assays were performed to determine which putative USF1 binding site was bound by USF1 in MCF-7 and MDA-MB-231 cells (below), IgG was used as a negative control. j Immunoblot analysis of USF1 and ESRP1 in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. k The correlation between USF1 and ESRP1 expression in breast cancer tissues from TCGA database was determined by Spearman correlation coefficients (r = 0.264, n = 1109, p < 0.001). Data were represented as means ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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