Fig. 1

CircRYK is upregulated by TGF-β1 in GBM. (A) Heatmap of all differentially expressed circRNAs after treatment of primary cells with 0 ng/ml or 10 ng/ml TGF-β1 for 24 h. (B) The expression levels of the three most highly expressed circRNAs were confirmed via qRT‒PCR. (C) qRT‒PCR was utilized to confirm the copy numbers of circRYK. (D) The expression of circRYK was analysed in ten normal brain tissues (NBTs), twenty low-grade gliomas (LGGs), and twenty high-grade gliomas (HGGs) by qRT‒PCR. (E) The splicing sequence and genomic position of circRYK are shown schematically. (F) Agarose gel electrophoresis was used to confirm the existence of the circRYK primer (175 bp). The “head to tail” splicing locations of circRYK are shown by the arrow. (G) A schematic representation of the structure of circRYK was generated via Sanger sequencing. (H) The reverse transcription studies employed random hexamers or oligo (dT)18 primers. qRT‒PCR was utilized to measure the relative RNA levels, and random hexamer primers were used as a standard. (I) After actinomycin D treatment, qRT‒PCR was used to calculate the relative quantities of RNA in U87 cells at specific time points. (J) The total RNA levels in U87 cells were evaluated via qRT‒PCR after treatment with RNase R or a control treatment. (K) The cellular localization of circRYK was examined utilizing cellular RNA fractionation methods. (L) FISH was utilized to analyze the cellular localization of circRYK. CircRYK is shown in green. Nuclei were stained with DAPI. Scale bar, 100 μm. Each experiment was performed three times, and the results are displayed as the mean ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001)