Figure 2.

circSnx5 Knockdown Promoted the Inflammatory Phenotype of DCs

DCs were transfected with a circSnx5 shRNA (shcircSnx5) or shRNA negative control (shNC) for 6 h before LPS treatment. (A) Schematic model of the shRNAs. shcircSnx5 targets the back-splice junction of circSnx5. (B and C) qRT-PCR for (B) circSnx5 and (C) Snx5 mRNA in DCs treated with shRNA as described above (n = 6 independent DC preparations). (D) DC subset treated with shRNA as described above was stained with the directly conjugated antibodies indicated and analyzed by flow cytometry (n = 4 independent DC preparations). (E) The supernatant was then harvested and analyzed for IL-12, IL-10, TGF-β, and IL-35 by ELISA (n = 4 independent DC preparations). (F–H) DCs were transfected with shcircSnx5 or NC for 6 h and then treated with 200 ng/mL LPS (12 h) and 10 μg/mL mitomycin C (2 h). (F) T cell proliferation was assessed by BrdU ELISA. (G and H) For Treg analysis, the mixed cells were stained with anti-CD4, anti-CD25, and then Foxp3 staining buffer and analyzed by flow cytometry (G) and the percentage of Tregs is shown in (H). n = 6 independent DC preparations. (I) qRT-PCR analysis for circSnx5 between shcircSnx5-transduced DCs and cells rescued by overexpression of circSnx5 (shcircSnx5-res). n = 6 independent DC preparations. (J) Co-stimulators (CD80, CD86, and MHC class II) were detected by flow cytometry in shcircSnx5-transduced or shcircSnx5-res DCs (n = 4 independent DC preparations). (K) Cytokine levels (IL-12, IL-10, TGF-β, and IL-35) were analyzed by ELISA in shcircSnx5-transduced or shcircSnx5-res DC supernatants (n = 4 independent DC preparations). Con, DCs treated with 200 ng/mL LPS for 12 h; NC, negative control; shcircSnx5, LPS-DCs were transfected with shcircSnx5. Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, by Student’s t test.