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Fig. 2 | Molecular Cancer

Fig. 2

From: A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1

Fig. 2

Circ-AKT3 encodes a 174aa novel protein termed as AKT3-174aa. a Upper panel, the putative ORF in circ-AKT3. Lower panel, the sequences of the putative ORF are shown. Note that the circ-AKT3 used an overlap start-stop codon. b The putative IRES activity in circ-AKT3 was tested. Upper panel, IRES sequences in circ-AKT3 or its different truncations/mutation were cloned between Rluc and Luc reporter genes with independent start and stop codons. Lower panel, the relative luciferase activity of Luc/Rluc in the above vectors was tested. Error bars represent three independent experiments, **, p < 0.01, ***, p < 0.001. c Illustration of AKT3-174aa sequence and AKT3 sequence. The antibody used in the study recognized both proteins. d AKT3 and AKT3-174aa expression were detected in established cell lines and several paired GBM samples. e U251 cells were transfected with empty vector, circ-AKT3 IRES mutated vector, circ-AKT3 vector and linearized AKT3 vector, respectively. Circ-AKT3, AKT3 and AKT3-174aa level were decided. Error bars represent three independent experiments, ***, p < 0.001. f Total proteins from circ-AKT3 or control plasmid-transfected U251 cells were separated via SDS-PAGE. AKT3-174aa overexpression was confirmed by immunoblotting. The differential gel bands between 26 kD and 34 kD was cut and subjected to LC-MS/MS. The identified AKT3-174aa amino acids are shown in red. g NHA cells were transfected with control shRNA or circ-AKT3 shRNAs. Circ-AKT3, AKT3 and AKT3-174aa level were decided. Error bars represent three independent experiments, ***, p < 0.001. h Flag tagged AKT3-174aa was transfected into U251 cells. Immunofluorescence staining using anti-Flag was performed to show the AKT3-174aa cellular localization. Scale bars, 20 μM. i Semi-quantitative analysis of AKT3-174aa expression level and GBM patient overall survival (OS) in the 38 patient cohort. **, p < 0.01. j Left, semi-quantitative analysis of AKT3 expression level and GBM patient overall survival (OS) in the 38 patient cohort. Right, semi-quantitative analysis of AKT3 expression level and GBM patient overall survival (OS) in TCGA data base

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