Journal: Nature Communications

Article Title: Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair

doi: 10.1038/s41467-022-34287-5

Figure Lengend Snippet: a Heatmap showing changes in expression levels of circRNAs >1.5-fold of Sham in TECs isolated from mice treated with mild- (20-min ischemia) or severe IRI (40-min ischemia) ( n = 3 mice in each group). See also Supplementary Fig. . b , c Expression of 8 circRNAs differentially expressed in RNA-sequencing between severe IRI and Sham mice, determined by quantitative RT-PCR (qRT-PCR) at day 1 ( b ) or day 14 ( c ) after IRI ( n = 5 mice in each group). d The RNA levels of circBNC2 and linearized BNC2 (lBNC2), examined by northern blots in various mouse tissues. e RT-PCR showing abundant circBNC2 in homogenates of normal kidney and liver, and downregulated expression of circBNC2 in human IRI-induced fibrotic kidney and in hepatitis B virus-induced liver fibrosis ( n = 3 in each group). f The expression of circBNC2, detected by northern blots, was abundant in mouse TECs and primary hepatocytes, and weak in mesangial cells (sv40mes13), podocytes (MPC-5) and primary stellate cells (HSCs). g The expression of circBNC2, detected by northern blots, was mainly observed in human TECs (HKC8 and HK2) and hepatocytes (L-02), and weak in mesangial cells (HMC), podocytes (HPC) and stellate cells (LX-2). h qRT-PCR showed that circBNC2 and lBNC2 expressed in the cytoplasmic of normal TECs (HK2). ACTB or U6 was the reference gene in cytoplasmic or nuclear part of TECs, separately. i Confocal FISH images showing cytoplasmic localization of circBNC2 in human TECs (HK2) and hepatocytes (L-02) cells. j The relative RNA levels of circBNC2 and lBNC2 assayed by qRT-PCR in HK2 treated with actinomycin D. k Northern blots using the Exon 2 probe (detecting circBNC2 and lBNC2) or junction-specific probe (detecting circBNC2) in total RNAs, in the presence or absence of RNase treatment. Total RNAs were extracted from HK2 cells. l qRT-PCR showing circBNC2 and lBNC2 expression in HK2 cells exposed to hypoxia or Aristolochic acids (AA) for 24 h. m , n qRT-PCR showing circBNC2 and lBNC2 expression in IRI- or AA-induced kidney fibrosis and CCl 4 -induced liver fibrosis in mice ( n = 6 in each group). o – q In situ hybridization showing circBNC2 expression in IRI- or AA-induced kidney fibrosis and CCl 4 -induced liver fibrosis ( o ) and the quantification data ( p , q ) ( n = 6 in each group). r – t In situ hybridization showing circBNC2 expression in IRI- or AA-induced human kidney fibrosis ( n = 4 in each group) and HBV-induced liver fibrosis ( n = 5 in each group) ( r ) and the quantification data ( s , t ). For h , j , l , n = 3 biologically independent cells. Data are expressed as means ± SD. Two-sided T -test was used for the comparison of two groups ( b , c , j , l , n , q , t ). One-way ANOVA with Bonferroni post hoc test was used for comparison among multiple groups ( m , p , s ). Source data are provided as a file.

Article Snippet: The primary antibodies used are as follows: anti-β-actin (Proteintech, cat. 20536-1-AP, dilution: 1:2000), anti-BNC2 (Novus, cat. NBP1-69078, dilution 1:1000), anti-αSMA (Cell Signaling Technology, cat. 19245, dilution 1:1000), anti-collagen I (Boster, cat. BA0325, dilution 1:1000), anti-FLAG (Sigma-Aldrich, cat. F1804, dilution 1:1000), anti-Fibronectin (Sigma-Aldrich, cat. SAB4500974, dilution 1:1000), anti-DHX9 (Abcam, cat. ab70777 or cat. ab26271, dilution 1:1000), anti-ADAR1 (Abcam, cat. ab168809, dilution 1:1000; Novus, cat. NBP2-92918, dilution 1:1000), anti-CDK1 (Cell Signaling Technology, cat. 9116, dilution 1:1000), anti-cyclin B1 (Cell Signaling Technology, cat. 12231, dilution 1:1000), anti-cyclin D1 (Cell Signaling Technology, cat. 55506, dilution 1:1000), anti-Lamin A/C (Cell Signaling Technology, cat. 4777, dilution 1:1000), anti-GAPDH (Cell Signaling Technology, cat. 5174, dilution 1:1000).

Techniques: Expressing, Isolation, RNA Sequencing Assay, Quantitative RT-PCR, Northern Blot, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization

Journal: Nature Communications

Article Title: Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair

doi: 10.1038/s41467-022-34287-5

Figure Lengend Snippet: 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. . 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 file.

Article Snippet: The primary antibodies used are as follows: anti-β-actin (Proteintech, cat. 20536-1-AP, dilution: 1:2000), anti-BNC2 (Novus, cat. NBP1-69078, dilution 1:1000), anti-αSMA (Cell Signaling Technology, cat. 19245, dilution 1:1000), anti-collagen I (Boster, cat. BA0325, dilution 1:1000), anti-FLAG (Sigma-Aldrich, cat. F1804, dilution 1:1000), anti-Fibronectin (Sigma-Aldrich, cat. SAB4500974, dilution 1:1000), anti-DHX9 (Abcam, cat. ab70777 or cat. ab26271, dilution 1:1000), anti-ADAR1 (Abcam, cat. ab168809, dilution 1:1000; Novus, cat. NBP2-92918, dilution 1:1000), anti-CDK1 (Cell Signaling Technology, cat. 9116, dilution 1:1000), anti-cyclin B1 (Cell Signaling Technology, cat. 12231, dilution 1:1000), anti-cyclin D1 (Cell Signaling Technology, cat. 55506, dilution 1:1000), anti-Lamin A/C (Cell Signaling Technology, cat. 4777, dilution 1:1000), anti-GAPDH (Cell Signaling Technology, cat. 5174, dilution 1:1000).

Techniques: Western Blot, Expressing, Immunoprecipitation, Quantitative RT-PCR, Transfection, Sequencing, Cell Cycle Assay, Flow Cytometry, Knock-Out, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Over Expression, Incubation

Journal: Nature Communications

Article Title: Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair

doi: 10.1038/s41467-022-34287-5

Figure Lengend Snippet: a , b IRES sequences in circBNC2 or its truncations were cloned between R-luc and F-Luc reporter genes ( a ) The predicted IRES activity in circBNC2 was tested by luciferase reporter assays ( b ). c Silver-staining of proteins from lysates of HK2 cells transfected with circBNC2 or empty vector. LC-MS analysis (samples from 3 independent experiments, n = 1 for each experiment) was used with the gel bands (70–100 kDa) from the lysates of HK2 cells overexpressing circBNC2. d Mass spectrometry result identifying a specific peptide sequence for circRNA translated BNC2 (ctBNC2). See also Supplementary Fig. . e , f Western blots showing ctBNC2 expression in HK2 cells exposed to hypoxia ( e ) or AA ( f ) for the indicated timepoints. g , h Western blots showing ctBNC2 expression in cortex homogenates from mice with IRI-induced kidney fibrosis ( g ) and in liver homogenates from mice with CCl 4 -induced liver fibrosis ( h ). i The proteins in lysates of HK2 cells precipitated with anti-ctBNC2 or anti-full-length BNC2 (BNC2-FL) were detected by immunoprecipitation using normal IgG as the negative control. The SDS-PAGE gel with abundant bands (25–35 and 55–70 kDa) were collected and analyzed by Mass spectrometry to identify proteins interacted with ctBNC2 or BNC2-FL (3 independent experiments, n = 1 for each experiment). j A Venn diagram showing the intersection presenting 6 proteins (CDK1, cyclin B1, RPS6, ATP5B, CCT2 and Fascin) bound to ctBNC2 in three replicated experiments. k , l CDK1 and cyclin B1 bound to ctBNC2, as shown by Mass spectrometry results. See also Supplementary Data . m , n Interaction of ctBNC2 with CDK1 and cyclin B1, as shown by western blotting following immunoprecipitation of either ctBNC2 ( m ) or CDK1 ( n ). o , p Interaction of CDK1 with cyclin B1 was inhibited in circBNC2-KO HK2 cells, as shown by western blotting following immunoprecipitation of either CDK1 ( o ) or cyclin B1 ( p ). q Interaction of CDK1 with cyclin B1 was increased by overexpressing circBNC2 in 24-h hypoxia-treated HK2 cells in a dose-dependent manner, as shown by Immunoprecipitation assay followed by western blot. r Western blots showing CDK1 and cyclin B1 nuclear translocation was inhibited in circBNC2-KO HK2 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. s Western blots showing CDK1 and cyclin B1 nuclear translocation in circBNC2 or circBNC2 no ATG overexpressed HK2 cells treated by hypoxia for 24 h. t To test the binding site of ctBNC2 with CDK1 and cyclin B1, deletion mutants of ctBNC2 were established and tagged with FLAG. See also Supplementary Table . u , v Interaction of CDK1, cyclin B1 with ctBNC2 truncated mutants in vitro as shown by western blotting following immunoprecipitation of FLAG. w , x Interaction of CDK1 with cyclin B1 was inhibited in circBNC2-KO L-02 cells, as shown by western blotting following immunoprecipitation of either CDK1 ( w ) or cyclin B1 ( x ). y Interaction of CDK1 with cyclin B1 was increased by overexpressing circBNC2 in 24-h hypoxia-treated L-02 cells in a dose-dependent manner, as shown by western blots. For b , o , p , q , w – y , n = 3 biologically independent cells. Data are expressed as means ± SD. Two-sided T -test was used for the comparison of two groups ( o , p , w , x ). One-way ANOVA with Bonferroni post hoc test was used for comparison among multiple groups ( b , q , y ). Source data are provided as a file.

Article Snippet: The primary antibodies used are as follows: anti-β-actin (Proteintech, cat. 20536-1-AP, dilution: 1:2000), anti-BNC2 (Novus, cat. NBP1-69078, dilution 1:1000), anti-αSMA (Cell Signaling Technology, cat. 19245, dilution 1:1000), anti-collagen I (Boster, cat. BA0325, dilution 1:1000), anti-FLAG (Sigma-Aldrich, cat. F1804, dilution 1:1000), anti-Fibronectin (Sigma-Aldrich, cat. SAB4500974, dilution 1:1000), anti-DHX9 (Abcam, cat. ab70777 or cat. ab26271, dilution 1:1000), anti-ADAR1 (Abcam, cat. ab168809, dilution 1:1000; Novus, cat. NBP2-92918, dilution 1:1000), anti-CDK1 (Cell Signaling Technology, cat. 9116, dilution 1:1000), anti-cyclin B1 (Cell Signaling Technology, cat. 12231, dilution 1:1000), anti-cyclin D1 (Cell Signaling Technology, cat. 55506, dilution 1:1000), anti-Lamin A/C (Cell Signaling Technology, cat. 4777, dilution 1:1000), anti-GAPDH (Cell Signaling Technology, cat. 5174, dilution 1:1000).

Techniques: Clone Assay, Activity Assay, Luciferase, Silver Staining, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Western Blot, Expressing, Immunoprecipitation, Negative Control, SDS Page, Translocation Assay, Binding Assay, In Vitro