Fig. 8

Homologous circEif3i in mouse performs similar physiological functions. A Schematic diagram of circEif3i generated from the Exon 5 and Exon 6 of Eif3i gene. B Sanger sequencing of head-to-tail splice junction in circEif3i. C The agarose gel electrophoresis was performed using the divergent primers and convergent primers to identify the existence of circEif3i. D qRT-PCR assay to determine circEif3i, Eif3i mRNA and Gapdh mRNA levels in samples after RNase R treatment. E qRT-PCR showed the stabilities of circEif3i and Eif3i mRNA in pancreatic cancer cell treated with 5 μg/ml actinomycin D (Act D) for 24 h. F qRT-PCR analyses of relative circEif3i in Panc02 cells transfected with sh-ctrl or sh-circEif3i. G Western blot showed the protein expression of p-SMAD3 (Ser423/425) and SMAD3 in Panc02 cells transfected with sh-ctrl or sh-circEif3i, with Tgf-β treatment (5 ng/ml, 15 min). H Representative images of the metastases (white arrows) of orthotopic models and HE staining of intestinal and mesentery metastatic nodules (Scale bar = 200 μm), and visible metastatic nodes were calculated (right panel). I Representative photographs of the liver metastasis model and HE staining of liver metastatic nodules with original magnification (Scale bar = 200 μm). The number of macroscopic metastases were calculated (right panel). J Representative images of immunohistochemistry analysis to measure the expression of p-SMAD3, MMP2, MMP9, and MMP14 in orthotopic tumours from negative control and circEif3i KD group (Scale bar = 50 μm). Data are shown as the mean ± SD of three replicates; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant