Fig. 3

Overexpression of circDYM inhibited microglial activation in vivo. a, b Effect of circDYM overexpression on iNOS levels in CUS (a) and LPS (b) models. Mice were microinjected with the circControl/circDYM-GFP lentivirus in the hippocampus. One week after microinjection, mice were exposed to a CUS protocol for 5 weeks, or they were intraperitoneally injected with LPS (1 mg/kg) for 5 successive days. Three representative immunoblots were presented from 6 mice/group. All data were presented as mean ± SEM. (CUS model, circDYM: F_(1,20) = 10.261, P < 0.01; CUS: F_(1,20) = 55.047, P < 0.001; interaction: F_(1,20) = 10.918, P < 0.01. LPS model, circDYM: F_(1,20) = 11.488, P < 0.01; CUS: F_(1,20) = 43.685, P < 0.001; interaction: F_(1,20) = 14.374, P < 0.01). c–g Effect of circDYM on microglial activation induced by CUS. Representative images of microglial immunostaining for Iba-1 in mice hippocampus, followed by 3D reconstruction and Sholl analysis (c). Scale bars: 200 μm (upper panel) and 100 μm (lower panel). Average soma size (d), branch number (e), total branch length (f), and total branch volume (g). All data were presented as mean ± SEM. n = 5 mice/group, 50 cells/group. (Average soma size, circDYM: F_(1,196) = 15.035, P < 0.001; CUS: F_(1,196) = 60.454, P < 0.001; interaction: F_(1,196) = 42.132, P < 0.001. Branch number, circDYM: F_(1,196) = 6.649, P < 0.05; CUS: F_(1,196) = 57.645, P < 0.001; interaction: F_(1,196) = 17.259, P < 0.001. Total branch length, circDYM: F_(1,196) = 60.587, P < 0.001; CUS: F_(1,196) = 73.005, P < 0.001; interaction: F_(1,196) = 38.486, P < 0.001. Total branch volume, circDYM: F_(1,196) = 47.866, P < 0.001; CUS: F_(1,196) = 127.414, P < 0.001; interaction: F_(1,196) = 67.190, P < 0.001). h–l Effect of circDYM on microglial activation induced by LPS. Representative images of microglial immunostaining for Iba-1 in mice hippocampus, followed by 3D reconstruction and Sholl analysis (h). Scale bars: 200μm (upper panel) and 100 μm (lower panel). Average soma size (i), branch number (j), total branch length (k), and total branch volume (l). All the data were presented as mean ± SEM. n = 5 mice/group, 50 cells/group. (Average soma size, circDYM: F_(1,196) = 50.359, P < 0.001; LPS: F_(1,196) = 69.865, P < 0.001; interaction: F_(1,196) = 23.044, P < 0.001. Branch number, circDYM: F_(1,196) = 7.061, P < 0.01; LPS: F_(1,196) = 63.546, P < 0.001; interaction: F_(1,196) = 10.267, P < 0.01. Total branch length, circDYM: F_(1,196) = 22.037, P < 0.001; LPS: F_(1,196) = 66.660, P < 0.001; interaction: F_(1,196) = 15.319, P < 0.001. Total branch volume, circDYM: F_(1,196) = 36.990, P < 0.001; LPS: F_(1,196) = 97.182, P < 0.001; interaction: F_(1,196) = 45.217, P < 0.001). **p < 0.01 and ***p < 0.001 versus circControl Control group; ^#p < 0.05 and ^###p < 0.001 versus circControl treated with CUS/LPS group