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. 2024 Jan 16;8:11. doi: 10.1038/s41698-024-00503-2

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a The putative open reading frame (ORF) in circCCDC7(15,16,17,18,19). Note that the circCCDC7(15,16,17,18,19) junction is inside the ORF. b The comparison of ID index score of internal ribosomal entrance site (IRES) among circCCDC7(15,16,17,18,19), circFBXW7 and circZNF609 using IRESfinder. c The relative abundance of circFBXW7, circITCH, circZNF609, circCCDC7(15,16,17,18,19) and GAPDH mRNA in PC3 cell lines detected by RT-qPCR after actinomycin D treatment at the indicated time points. The relative expression was normalized to the expression of 0 h. d Quantitative analysis of the expression of circCCDC7(15,16,17,18,19) between nuclear and cytoplasm in PC3 cells. e Gel image of RT-qPCR product of the circFBXW7, circCCDC7(15,16,17,18,19), circITCH, and GAPDH in ribosome enriched RNA. Mixed ribosome RNA from LNCaP, C4-2, PC3 and DU145 were used. f Upper panel: the sequence of the putative ORF is shown in black font with white background, internal start codon is shown with green font, internal stop codon is shown in red font, and the IRES sequence is shown in black word with gray background. Lower panel: the predicted amino acid sequence of circCCDC7-180aa. The amino acids with green background are the same to the linear CCDC7 and the last seven amino acids are novel. g FLAG tag antibody was used to detect circCCDC7-180aa expression in 293 T cells transfected with vector or overexpression plasmid. h CircCCDC7-180aa was confirmed by immunoblotting after IP. The circCCDC7-180aa were extracted and subjected to mass spectrometry and junction-specific peptide (SQESSTSGN) was identified (right panel). i Immunofluorescence assay demonstrated that circCCDC7-180aa was mainly located around nucleus in HCT116 cell line. GFP plasmid and FLAG tagged circCCDC7(15,16,17,18,19) or empty vector were co-transfected. Upper is GFP+vector; Lower GFP + FLAG tagged circCCDC7(15,16,17,18,19)_. j Western blot showed circCCDC7-180aa specifically enriched in membrane fraction. k ELISA assay demonstrated that circCCDC7-180aa is secreted into cell culture medium 2 days after plasmid transfection in 293 T cells. ***p < 0.001, ****p < 0.0001.