Fig. 2

Circ000–4585 promotes autophagy and metastasis of PCa cells in vitro and in vivo. A Schematic representation of the sites of the siRNA specific to the back-splice junction of circ_0004585. B Real-time qPCR analysis of the expression of circ_0004585 in PC-3 and DU145 cells with stable overexpression or silencing. C Autophagic flux was monitored in stable overexpressed and downregulated circ_0004585 PC-3 cells expressing endogenous LC3BII/I tagged with tandem fluorescent-mCherry-GFP as a reporter. GFP and mCherry signal colocalization (yellow dots) indicated the lack of phagophore or autophagosome fusion with lysosomes (Scale bar,50 μm). D Transmission electron microscopy (TEM) revealed the number of double-membrane autophagosomes in stable overexpressed and downregulated circ_0004585 PC-3 cells (Original magnification, × 1000, × 1600, respectively). E HE stain assay showed that circ_0004585 knockdown compromised aggressive characteristics of circ_0004585 upregulated PC-3 cells, and inhibited in vivo pulmonary metastases (n = 5 per group, Original magnification, × 10, × 100, respectively). F Representative image of metastatic lung colonization in nude mice (n = 5 per group). G Kaplan–Meier curves for nude mice. Bar graphs show the statistical analysis of three independent experiments (n = 5 per group, * P < 0.05; ** P < 0.01)