Fig. 1

TIF1γ and circPTK2 are down-regulated during TGF-β-induced EMT in NSCLC cells. a A549 cells underwent epithelial-mesenchymal transition (EMT) after TGF-β1 (5 ng/ml) treatment for 24 h. Cell morphology was observed and photographed with a phase-contrast microscope (upper). Scale bar, 50 μm. The expression of EMT-related makers including E-cadherin, N-cadherin and Vimentin (bottom left), and TIF1γ protein (bottom right) were examined by western blot. β-actin was used as internal control. Densitometry values for each protein were normalized to β-actin and shown below the corresponding bands. b RNA from epithelial and mesenchymal A549 cells were subjected to Arraystar Human circRNA Array analysis as described in Methods. Hierarchical cluster analysis (heat map) of microarray data was used to show the significant expression of circRNAs when comparing mesenchymal cells with epithelial cells (left). Red and green denoted high and low expression, respectively. Each column represents a test sample and each row represents a circRNA. Each group (treated with TGF-β1 for 0 h or 24 h) was analyzed in triplicate. In a zoomed-in view of partial (right), the expression of circPTK2 (hsa_circRNA_104703) was indicated as an arrow. c The sketch of genomic locus of circPTK2 in PTK2 gene. The expression of circPTK2 (circBase ID: hsa_circ_0008305) was validated by RT-PCR followed by sanger sequencing. Red arrows represent divergent primers, which are used to amplify the genome region of circPTK2 containing the back-splice junction site (JCT). d In A549 or H226 cells, divergent primers amplify circPTK2 JCT in cDNA but not in genomic DNA (gDNA), convergent primers amplify both circPTK2 JCT and linear PTK2 Exon 9. GAPDH was used as linear control. Red and black arrows represent divergent and convergent primers, respectively. Divergent primers spanning circPTK2 JCT yield a product of 110 bp, while the convergent primers amplifying PTK2 exon 9 yield a product of 141 bp. e Endogenous circPTK2 expression in A549 cells was validated by northern blots. RNase R was used to digest linear RNA. f Representative image of RNA fluorescence in situ hybridization for endogenous circPTK2 in A549 cells. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. g qRT-PCR analysis of circPTK2 expression in A549 and H226 cells treated with TGF-β1 for 24 h. Relative circPTK2 expression was determined with normalization against β-actin. h, i qRT-PCR analysis of miR-429/miR-200b-3p expression levels in A549 and H226 cells treated with TGF-β1 for 24 h. U6 was used as internal control. *P < 0.05; **P < 0.01