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. Author manuscript; available in PMC: 2022 Jul 19.
Published in final edited form as: Nature. 2021 Oct 20;599(7884):308–314. doi: 10.1038/s41586-021-04009-w

Extended Data Fig. 6. EccDNAs are potent immunostimulants for BMDMs.

Extended Data Fig. 6.

a, Confirmation of BMDMs identify by flow cytometry. After differentiating bone marrow cells with L929 conditioned medium for 7 days, cells debris (left) were first excluded and singlets (middle) then phenotyped on the basis of F4/80 and CD11b expression (right) to define BMDM, the numbers indicate the percentage of gated cells. Data were further analyzed with FlowJo. V10.8.0 and shown as representative of 2 independent experiments.

b-c, EccDNAs induce cytokine genes in BMDMs. Bar graphs showing the induction of mRNA (b) and protein (c) of IL-6 and TNF-α in BMDMs that transfected with varying levels of eccDNA, compared with fragmented linear DNA, and poly(dG:dC). RT-qPCR were performed after 12-hours transfection. ELISA analysis (c) was performed after 24-hours transfection. Data is shown as mean ± S EM of replicates (n=4 per group) of a representative experiment of three independent experiments. Comparisons were performed on biological replicates with equal amount of DNA transfection by Ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significant; nd, not detected.