Fig. 5

Upregulation of circMETTL9 in cultured astrocytes promotes inflammatory signaling. A Representative image showing co-localization of the astrocyte marker GFAP (green) and circMETTL9 (red) in rat cerebral cortex. White arrows show the typical co-localization of circMETTL9 and GFAP in the injured cortex. Scale bar = 50 µm. B LPS-induced upregulation of circMETTL9 in cultured astrocytes as measured by qPCR. *P < 0.05 by one-way ANOVA. C Peak expression after a 12-h treatment with 500 ng/mL LPS. ***P < 0.001 by one-way ANOVA. D, E Design of siRNA sequences for circMETTL9 knockdown and detection of circMETTL9 and METTL9 mRNA expression levels following transfection for 48 h using Lipo3000. *P < 0.05 and **P < 0.01 by Student’s t-test. F–J Changes in the astrocytic expression levels of pro-inflammatory cytokines CCL2, CXCL1, CCL3, CXCL3, and CXCL10 after circMETTL9 knockdown. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA. (K) CircMETTL9 is significantly upregulated after 48 h of plasmid transfection. *P < 0.05, Student’s t-test. L–P Changes in expression levels of CCL2, CXCL1, CCL3, CXCL3, and CXCL10 in astrocytes overexpressing circMETTL9 compared to controls. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA