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Fig. 4 | Molecular Cancer

Fig. 4

From: circPTEN1, a circular RNA generated from PTEN, suppresses cancer progression through inhibition of TGF-β/Smad signaling

Fig. 4

CircPTEN1 suppresses TGF-β-mediated CRC metastasis. A. Identification of the circPTEN1-protein complex pulled down by the circPTEN1 junction probe with protein extracts from LoVo cells. The arrow indicates the band containing Smad4. B. RIP assays showing the association of Smad4 with circPTEN1. Left, IP efficiency of Smad4 antibody shown by western blot. Right, relative enrichment representing RNA levels associated with Smad4 relative to an input control. IgG served as a control. C. Colocalization analysis of Smad4 and circPTEN1 using protein IF and RNA FISH assays, respectively. Scale bar, 10 μm. D and E. The indicated cells were treated with 5 ng/mL TGF-β1 for 48 h, and motility was sequentially evaluated through Transwell migration assays and invasion assays. (D) Representative images. Scale bar, 40 μm. (E) Migrated cells were counted in five random fields per well. Upper, DLD1. Lower, LoVo. RS: pLCDH-circPTEN1-RS. ****, p < 0.0001. F. Cells treated with TGF-β1 as indicated in Fig. 4D were subjected to the 3D multicellular tumor spheroids invasion assay. Upper, representative images. Lower, the invading area of 15 cells was analyzed per group. ****, p < 0.0001. The scale bars represent 600 μm. RS: pLCDH-circPTEN1-RS. G and H. Increased or decreased tumor metastasis formed in the lungs of mice through vein tail injection (G) or in the livers of mice through inferior hemispleen implantation (H) of circPTEN1 knockdown cells and circPTEN1 overexpression cells treated with TGF-β1 as indicated in Fig. 4D. Left panel, representative bioluminescent images. Right panel, statistical analysis of bioluminescent tracking plots. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001

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