Extended Data Fig. 7: The circularity of eccDNAs, but not the sequence, is critical for their immunostimulant activity.

a, Bar graphs showing the relative IL-6 and TNF-α mRNA levels. Data is shown as mean ± s.e.m. of replicates (n=4 per group) of a representative experiment in three independent experiments. b, c, Bar graphs showing the relative IL-6 and TNF-α mRNA levels (b), and protein (ELISA) levels (c). Data is shown as mean ± s.e.m. of replicates (n=4 per group) of a representative experiment in three independent experiments. d, Diagram of DNA transfection efficiency and stability assay. Step-1: 5 Phosphorothioate (purple “*”) end-protected synthetic linear DNA (PS-syn-linear) and its circular form with equal number of phosphorothioate bonds (PS-Syn-circular) were transfected to BMDCs in the same way as in Fig. 5 for either 1 or 12 h. Step-2: cells were lysed in 100 μl lysis buffer and treated with Thermoliable Proteinase K to prepare total DNA. Step-3: 4 μl total DNA was used for qPCR with a set of primers that amplify a 117 bp fragment in both linear and circular DNA. e, qPCR analysis of the samples prepared as described in (d). Transfected DNA level was normalized to that of 10 ng/ml PS-syn-linear transfection (n= 4 or 6, as indicated by dotes). Bars indicate mean ± s.e.m. of three independent experiments. f, 12 h after transfection of indicated DNA at 30 ng/ml, medium was collected for ELISA essay, Data are shown as mean ± s.e.m. of replicates (n=4 per group) of a representative experiment in three independent experiments. Comparisons in a–c were performed on biological replicates by Ordinary one-way ANOVA with Tukey’s multiple comparison test; two-tailed unpaired t tests (e, f). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significant.