Fig. 3

Elevated expression of circMETTL9 in cortex following TBI and successful siRNA-mediated knockdown. A Gross observation of brain tissue in TBI rats. Left panel: shaded areas illustrate the injured cortex that was harvested for qPCR and WB analysis. Right panel: the microphotographed areas used in IF, FISH, and TUNEL. Scale bar = 3 mm. B Expression levels of circMETTL9 in cortex on days 1, 3, 7, and 14 post-TBI as measured by qPCR. Expression peaked on day 7. **P < 0.01 and ***P < 0.001 by one-way ANOVA. C Expression of METTL9 mRNA in cortex of TBI model rats vs. sham controls. D Left panel: FISH image indicating that circMETTL9 (red) was highly expressed at the injury site of TBI model rats. Scale bar = 50 µm. Right panel: quantification of mean fluorescence intensity of circMETTL9 (n = 3 per group). **P < 0.01 by Student’s t-test. E Experimental procedure and timeline for in situ knockdown experiments. F Representative images of rat cerebral cortex microinjected with AAV2/9 virus expressing siRNA and GFP. GFP expression was measured 4 weeks after microinjection as an index of viral vector expression. Scale bar = 100 µm. G, H Expression levels of circMETTL9 and METTL9 mRNA 4 weeks after microinjection as measured by qPCR. Expression of circMETTL9 was significantly downregulated while METTL9 mRNA was not significantly altered (n = 6 per group). ****P < 0.0001 by Student’s t-test