Overexpression constructs for visualization of exogenous circRNAs tagged with Pepper. (A) Graphical representation of the p-circ overexpression plasmids designed to tag and visualize circRNAs with the fRNA Pepper. Green and blue arrows represent forward primers and yellow arrows represent common reverse primers, respectively, used to amplify the BSJ of circ-HDGFRP3 and the linear isoforms. (B) Expression levels quantified through qPCR of circ-HDGFRP3 and of the linear isoform upon p-circ-Ex4 and p-circ-Ex5 transfection, with (+) or without (−) doxycycline administration, normalized on GAPDH levels. Error bars represent ± SEM (N = 3). (*) P = 0.05 corresponds to two-tailed unpaired Student's t-test (N = 3) calculated on DDCt values normalized on the -rt samples to account for plasmid DNA contamination. (C) Nuclear/cytoplasmic fractions of exogenous circ-HDGFRP3 and its relative linear isoform expressed in percentage. GAPDH and preGAPDH were used as cytoplasmic and nuclear controls, respectively. Error bars represent ± SEM (N = 3). (D) Left chart represents normalized fluorescence intensities fluctuations of HBC620 dye bound to ROMO1-4xPepper (gray, solid line) or to p-circ-Ex4 transcript (magenta, solid line) upon 120, 180, and 240 min of RNase R treatment. Cells transfected with overexpression plasmids but not treated with Triton X-100, hence not exposed to RNase R, were used as a negative control (dashed lines). Error bars represent ± SEM. (***) P ≤ 0.001 corresponds to two-tailed unpaired Student's t-test (N = 3) calculated on unnormalized fluorescent intensity measurements of treated versus nontreated cells at 240 min. Right chart represents RNA levels of ROMO1 mRNA (gray), circ-HDGFRP3 (magenta) and of the linear isoform (black) exposed (solid line) or not exposed (dashed line) to 120, 180, and 240 min of RNase R treatment. Error bars represent ± SEM. (**) P ≤ 0.01 corresponds to two-tailed unpaired Student's t-test (N = 3) calculated on DCt of treated versus nontreated RNAs at 240 min. (E) Representative cells transfected with ROMO1-4xPepper (left panel) or p-circ-Ex4 (right panel) constructs treated and nontreated with RNase R at 0, 120, 180, and 240 min time points. Yellow arrows highlight focal foci in cells transfected with p-circ-Ex4. Scale bars, 5 μm.
