Fig. 2

circINSIG1 encodes a 121 amino acid protein. A Upper panel, the putative open reading frame (ORF) in circINSIG1. Lower panel, the sequences of the putative ORF encoded amino acid sequences are shown. B Polysome profiling in cytoplasmic extracts of HCT8 cells were fractionated through sucrose gradients, and the relative expression of circINSIG1, INSIG1 mRNA and circCAMSAP1 were analyzed by qRT-PCR in the gradient fractions. INSIG1 mRNA served as a positive control. circCAMSAP1 served as a negative control. C Western blot analysis of the expression of circINSIG1-121 with Flag antibody and circINSIG1-121 antibody. D The lysates from HCT8 cells with circINSIG1 overexpression were separated by SDS-PAGE. Protein bands near 14 KD were excised manually and summited for identification by LC–MS/MS. E The identified circINSIG1-121 unique peptide sequences. F Immunohistochemistry (IHC) images of circINSIG1-121 and INSIG1 expression in CRC tissues (left) and semiquantitative scoring of circINSIG1-121 and INSIG1 expression in 20 CRC tissues (right). G Western blot analysis of circINSIG1-121 expression in 10 paired CRC samples and normal adjacent tissues. H Kaplan-Meier curves for survival of CRC patients with low vs. high expression of circINSIG1-121. I Kaplan-Meier curves for survival of CRC patients with low vs. high expression of INSIG1. J Kaplan-Meier curves for survival of CRC patients with INSIG1 levels in the TCGA database. N, normal adjacent tissues; T, tumor tissues. Values are represented as mean ± SD, by 2-tailed Student’s t test