Fig. 1: Characterization of circTGFBR2(3-6), an activator of TGF-β/SMAD signaling.

A Diagram showing the results of shRNA-mediated screening of TGFBR1 or TGFBR2 pre-mRNA-derived circRNAs in MDA-MB-231 cells stably expressing a SMAD3/4-driven (CAGA)₁₂-dynGFP reporter in the presence of TGF-β (0.5 ng/mL). The x- and y-axes represent relative reporter activity from two independent experiments. shRNAs targeting linear TGFBR1 and TGFBR2 mRNA were taken along as control. B Illustration of circTGFBR2(3-6) biogenesis from TGFBR2 pre-mRNA. Sanger sequencing confirmed the back-splicing junction (BSJ) sequence of circTGFBR2(3-6). C PCR analysis of circTGFBR2(3-6) amplification from genomic DNA (gDNA) and complementary DNA (cDNA) of MDA-MB-231 and MCF10A-M2 cells, visualized by agarose gel electrophoresis. The schematic shows the positions and orientations of convergent and divergent PCR primers. D The workflow for preparing the MDA-MB-231-derived enriched circRNA pool (ECP) is illustrated on the left. The full-length circTGFBR2(3-6) PCR product, amplified from cDNA of the ECP, was analyzed by agarose gel electrophoresis (shown on the right). E RT-qPCR analysis of circTGFBR2(3-6) and TGFBR2 mRNA expression in MDA-MB-231 cells following RNase R treatment. Data present mean ± SEM from three biological replicates. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. F Subcellular localization analysis of circTGFBR2(3-6) in MDA-MB-231 cells by RT-qPCR. Long non-coding RNA LETS1 [96] and GAPDH mRNA serve as nuclear and cytoplasmic markers, respectively. Data are presented as mean ± SEM from three biological replicates. G In situ hybridization analysis of circTGFBR2(3-6) subcellular localization using a probe specifically targeting its BSJ sequence in MDA-MB-231 cells. Scale bar = 6.22 μm. Cells and nuclei are outlined in black, and red arrows indicate circTGFBR2(3-6) signals.