Figure 2.

Partial truncation of intronic sequences increases circRNA expression

(A) Portions of the HIPK3 left and right introns were deleted; deletions are numbered from the start of their respective intron. (B–D) Constructs were transfected into HEK293 cells and expression assayed at 4 days post-transfection by (B) GFP fluorescence, (C) western blot analysis, with actin as a loading control (quantification below), and (D) northern blot analysis, probing for GFP sequences (quantification below). (E) Either the left or right partial Alu element was deleted from the HIPK3 introns. (F and G) Constructs were transfected into HEK293 cells and expression assayed at 4 days post-transfection by (F) western blot analysis, with actin as a loading control, and (G) northern blot analysis, probing for GFP sequences. On northern blots, the asterisk refers to an additional circular band. Western and northern blots were quantified as detailed in Materials and methods and graphed relative to the unchanged HIPK3 intron construct. Student’s t test was performed to test for statistical significance. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.00005.