Fig. 1

Identification of circWSB1 in BC cells in response to hypoxia. a Western blot analysis of HIF1α in MCF-7 cells cultured under normoxia or hypoxia for 48 h. b Volcano plot of the differentially expressed circRNAs between MCF-7 cells treated with normoxia and hypoxia for 48 h. The blue dots and red dots represent downregulated and upregulated circRNAs with statistical significance, respectively. c and d Heatmaps of 20 most increased and decreased mRNAs (c) and eight circRNAs derived from WSB1 (d) between normoxic and hypoxic MCF-7 cells. e RT-PCR products of hsa_circ_0042496, hsa_circ_0042489, hsa_circ_0042493 and hsa_circ_0007716 detected by agarose gel electrophoresis. f qRT-PCR analyses of the relative expression of hsa_circ_0042496, hsa_circ_0042489, hsa_circ_0042493 and hsa_circ_0007716 in MCF-7 cells cultured under normoxia and hypoxia. g Relative expression of hsa_circ_0007716 detected by qRT-PCR in multiple BC cell lines treated with normoxia and hypoxia. h Schematic diagram illustrating the generation of hsa_circ_0007716 from its host gene WSB1. i Sanger sequencing of back-splicing junction of hsa_circ_0007716 (circWSB1). j circWSB1 and β-actin were amplified from gDNA and cDNA of MCF-7 cells using both convergent and divergent primers, respectively, followed by agarose gel electrophoresis. k qRT-PCR analysis to determine the relative level of circWSB1 and WSB1 mRNA in MCF-7 cells treated with or without Rnase R at 37 ℃ for 30 min. l Relative expression of circWSB1 and WSB1 mRNA in MCF-7 cells treated with or without actinomycin D (5 μg/mL) was examined by qRT-PCR. m Cytoplasmic/Nuclear fractionation followed by qRT-PCR indicating the subcellular localization of circWSB1 in MCF-7 cells cultured under hypoxia. Data are shown as mean ± SD and representative of three independent experiments in (f-g) and (k-m). *P < 0.05, **P < 0.01, ***P < 0.001