(A) HeLa cells were transfected with 500 ng of DNA plasmid programming ZKSCAN1 or td introns that produce a linear RNA or circRNA encoding GFP-IRES. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3); *p < 0.05 throughout.
(B) CircRNA produced from Drosophila laccase2 introns flanking GFP-IRES exon does not stimulate innate immune genes. HeLa cells were transfected with 500 ng of DNA plasmid that produce a linear RNA or circRNA encoding GFP-IRES directed by laccase2 introns.
(C) Co-expression of programmed protein-assisted and autocatalytic-splicing td circRNA still stimulates innate immune genes. HeLa cells were transfected with 500 ng of DNA plasmid programming ZKSCAN1 intron or sequential ZKSCAN1 and td introns encoding circRNA GFP-IRES. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, to confirm both the autocatalytic-splicing and protein-assisted splicing processes occur. Means ± SEM are shown (n = 3).