Fig. 2: Downregulation of circBNC2 promotes epithelial cell G2/M arrest after injury.

a Western blots showing DHX9 expression in lysates from HK2 cells exposed to hypoxia or AA for 24 h. b, c The primer sets designed in the pre-mRNA of BNC2 precursor (b) and the transcript abundance of amplicons a-d relative to input, detected by RNA immunoprecipitation with anti-DHX9 in lysates from HK2 cells treated with hypoxia for 24 h, followed by qRT-PCR assay (c). d qRT-PCR showing DHX9, circBNC2, lBNC2 and pre-mRNA of BNC2 (pmBNC2) expression in 24-h hypoxia-treated HK2 cells transfected with siRNAs targeting DHX9. e The top 10 significantly enriched Gene Ontology (GO) terms of the differentially expressed genes in mRNA sequencing of circBNC2-KO HK2 cells compared to wild-type (WT). f–h Cell cycle analysis by flow cytometry in HK2 cells, showing knockout of circBNC2 induced G2/M cell cycle arrest (f, g), especially G2 phase cell (h). i, j Immunofluorescence staining for p-H3 in circBNC2-KO HK2 cells showing increase in G2 phase positive cells (i) and the quantification data (j). See also Supplementary Fig. 2n. k, l Western blots showing cyclin B1 and cyclin D1 expression in circBNC2-KO HK2 cells (k) and the ratio of cyclin B1/cyclin D1 (l). m, n Secretion of TGF-β1 (m) and CTGF (n) by circBNC2-KO HK2 cells was examined by ELISA. o qRT-PCR showing mRNA levels of αSMA, COL1A1, FN, TGFB1 and CTGF expression in circBNC2-KO HK2 cells. p, q Western blots showing protein levels of αSMA, Col I, and FN in circBNC2-KO HK2 cells (p), and the quantification data (q). r The top 10 significantly enriched Gene Ontology (GO) terms of the differentially expressed genes in mRNA sequencing of circBNC2-KO L-02 cells compared to wild-type. s–u Cell cycle analysis by flow cytometry in L-02 cells showing knockout of circBNC2 induced G2/M cell cycle arrest (s, t), especially G2 phase cell cycle arrest (u). While overexpression of circBNC2 partially rescued the G2/M cell cycle arrest in circBNC2-KO cells. v Levels of TGF-β1 in supernatants of circBNC2-KO L-02 cells, examined by ELISA. w–y Western blots showing protein levels of αSMA and Col I in LX-2 cells incubated with conditional medium (C.M.) from circBNC2-KO L-02 cells or WT L-02 cells for 24 or 48 h. For c, d, g, h, j, l–o, q, t–v, x, y, n = 3 biologically independent cells. Data are expressed as means ± SD. Two-sided T-test was used for the comparison of two groups (c, h, j, l, m, n, o, q, u, v, x, y). One-way ANOVA with Bonferroni post hoc test was used for comparison among multiple groups (d, g, t). Source data are provided as a Source Data file.