Fig. 1

CircItgb5 is upregulated by PDGF-BB stimulation in PASMCs. A Heatmap showing a subset of circRNAs differentially expressed in PASMCs treated with PDGF-BB (100 ng/ml) for 12 h using high-throughput circRNAs sequencing. C, control; B, PDGF-BB. Fold change > 2, FKPM > 0.5 and FDR < 0.05. The colors in the heatmap represent fold change. B Validation of top 22 upregulated circRNAs from circRNAs-seq. RT-qPCR was conducted following PDGF-BB treatment for 12 h. C, control; B, PDGF-BB. Fold change > 2, FDR < 0.05. The top five upregulated circRNAs were highlighted with red frame. The colors in the heatmap represent fold change. C Expression of the top five upregulated circRNAs and the corresponding linear RNAs were detected by RT-qPCR following RNase R treatment. C, circular; L, linear. *0.01 ≤ P ≤ 0.05 versus Mock. D The presence of circItgb5 was detected by RT-PCR in PASMCs. Divergent primers amplified circItgb5 from cDNA, but not from gDNA. GAPDH was used as a positive control. C, control; B, PDGF-BB; cDNA, complementary DNA; gDNA, genomic DNA. E Schematic illustration showing exon 9 to 10 of Itgb5 circularization to form circItgb5 (black arrow). The sequence of circItgb5 was analyzed by Sanger sequencing. F The FISH assay was conducted to detect circItgb5 in PASMCs using Cy3-labeled probes (circItgb5). Nuclei were stained with DAPI. α-SMA is a characteristic marker of PASMCs. DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride. Original magnification, × 400; Scale bar = 50 μm. G RT-qPCR was conducted to detect circItgb5 expression in PA of MCT-treated rats and hypoxia-treated rats. PA, pulmonary arteries. n = 6. **0.001 ≤ P ≤ 0.009 versus control, ^##0.001 ≤ P ≤ 0.009 versus normoxia. All data are presented as means ± SD (n = 3 biological replicates)