Fig. 2

Circ-SMO encodes SMO-193a.a. a 293T cells transfected with circ-SMO or circ-SMO noATG plasmid were subjected to polysome profiling assay. Left, Representative polysome profile. Cell lysates were fractionated to collect non-ribosome fractions (N), monosome (M), light polysome (L), and heavy polysome (H) by 5–50% sucrose gradient centrifugation. Dashed lines indicate collected fractions. Right, Detection of circ-SMO and SMO mRNA in indicated fractions by RT-qPCR in 293T cells with indicated modifications. b Left, Predicted start and stop codon of ORF in circ-SMO. Right, Predicted amino acid sequences of SMO-193a.a. and antigen sequences for SMO-193a.a. antibody. c Identification and activity test of internal ribosomal entry site (IRES) in circ-SMO using circular vector-based luciferase reporter assay. EMCV-IRES was positive control. d Immunoblot (IB) and mass spectra (MS) identification of SMO-193a.a. in 373 GBM cells with circ-SMO overexpression (OV) and in 3691 CSC. e Upper, Illustration of SMO shRNAs and circ-SMO shRNAs. Lower, SMO and SMO-193a.a. expressions were detected using SMO-193a.a. polyclonal antibody in 3691 CSC treated with indicated shRNAs. f Left, Illustration of endogenous circ-SMO, circ-SMO overexpression vector, splicing donor site mutant vector (mutSD), circularization frame deletion vector (circ-frame Del), circ-SMO-3XFlag vector, and linearized SMO-193a.a.-3XFlag vector. Right, IB of cells OV with above described different vectors using anti-SMO-193a.a. antibody or Flag antibody. g. Left, Representative immunofluorescence (IF) images of cells OV with indicated vectors and stained with anti-Flag antibody. Bars, 5 μm. Right, Live image of SMO-193a.a.-mCherry in 373 GBM cells. Bars, 5 μm. h Left, IB of several established cell lines using anti-SMO-193a.a. antibody. Right, IB of seven random selected GBM samples and adjacent NB using anti-SMO-193a.a. antibody. i Upper, semi-quantification of SMO-193a.a. expression based on immunoblot band intensity relative to beta-tubulin in a previously described cohort of 86 GBM samples and paired NB. Lower, Survival analysis was conducted based on SMO-193a.a. expression in 86 GBM patients. The median score of relative expression levels in tumor tissues based on semi-quantification was used as the cutoff to define “high” or “low” SMO-193a.a. expression. MST, median survival time. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from at least three experiments with similar results