Supplementary Figure 3: Overexpression of circRNA and effects of EIciRNA or mRNA knockdown.

(a) Gel images show the size of the RT-PCR products with the divergent primers. Except for the vector control and the circ-Exon constructs, the overexpressed circRNAs were also intron retaining examined with the “primer set 1”. The RT-PCR bands show the size of the PCR product, and the band intensity does not represent relative expression levels. The junction in the circRNA produced by the circEIF3J_1 kb plasmid was different from that of circEIF3J, and this difference is shown in the gel image and sequence diagram below. (b) Knockdown of circEIF3J or circPAIP2 with the siRNA resulted in a decrease in the parental gene mRNA level in HEK293 cells. (c) ASO sequences for the knockdown of circEIF3J and circPAIP2. circEIF3J ASO was used to target the intron contained in circEIF3J, and a control ASO targeted intron 4 in the EIF3J gene; circPAIP2 ASO targeted the junction of circPAIP2, and a scrambled ASO was used as a control. Sequences in black indicated DNA, and sequences in green indicate RNA. (d) Knockdown of circEIF3J had no effect on the mRNA level of PAIP2 and CTDSPL2 (the 5’ neighbouring gene of EIF3J); knockdown of circPAIP2 had no effect on the mRNA level of EIF3J or MATR3 (the 5’ neighbouring gene of PAIP2). (e) siRNA knockdown efficiency of circEIF3J and circPAIP2 for the experiments shown in Fig 3c. (f) Nuclear run-on experiments showed that mRNA knockdown had no effect on the transcription of the indicated genes. siRNA knockdown efficiency of EIF3J or PAIP2 mRNA is shown below the nuclear run-on data. All data were from triplicate experiments. Error bars represent the s.e.m. **, P value < 0.01. P values were determined with two-tailed Student’s t-test.