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. 2017 May;23(5):619–627. doi: 10.1261/rna.056838.116

FIGURE 2.

FIGURE 2.

Csy4 activates circGFP-CD reporter expression to switch from dsRed to GFP. (A) Representative images of dsRed (top) and GFP (bottom) fluorescence from HEK293 cells expressing circGFP (far left), or circGFP-CD in the absence of Csy4 (middle left), presence of native Csy4 (middle right), or presence of Csy4-H29A (far right) 72 h post-transfection. Schematics of major pre-mRNA species present before splicing are depicted above each column, and schematics of the major RNA species post-splicing are depicted on the left of each row. The dsRed (B) and GFP (C) fluorescence were quantified from images taken 72 h post-transfection of HEK293 cells using the FIJI image processing package (Schindelin et al. 2012). Error bars indicate standard deviation of integrated density calculated from nine images from three biological replicates (three images per replicate). Statistical significance was calculated using a two-tailed Student's t-test ([****] P ≤ 0.0001, [*] P ≤ 0.05). (D) Western blot detecting GFP or loading control Actin for lysates of HEK293 cells transfected as indicated.