Fig. 2

Characterization and clinical features of circCCDC9. a. We confirmed the head-to-tail splicing of circCCDC9 in the circCCDC9 RT-PCR product by Sanger sequencing and also determined its genomic size and sequence as reported in the circBase database. b. RT-PCR validated the existence of circCCDC9 in MKN45 and AGS cell lines. CircCCDC9 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. c and d. The expression of circCCDC9 and CCDC9 mRNA in both MKN45 and AGS cell lines was detected by PCR assay followed by nucleic acid electrophoresis or qRT-PCR in the presence or absence of RNase R. e. FISH assay showed that circCCDC9 was predominantly localized in the cytoplasm. Nuclei were stained with DAPI for blue color, and cytoplasmic circCCDC9 were stained for red color. (magnification, 400×, scale bar, 100 μm) f. Kaplan-Meier survival curves of GC patients with low and high circCCDC9 expression. Using median circCCDC9 value as a cutoff. Data were showed as mean ± SD; ns indicated no significance. **P < 0.01, ***P < 0.001