Fig 2: Protein levels of BMAL1 in brain tissues after ICH in rats. (A) Western blot analysis of the relative BMAL1 expression levels in the rat brain tissues at various time points. (B) Quantification of the BMAL1 protein levels at various time points. (C) Double immunofluorescence staining was performed in brain sections using antibodies against BMAL1 (green) and a neuronal marker (red). Nuclei were fluorescently labeled with DAPI (blue). Arrow heads indicate BMAL1/NeuN-positive cells. (D) Double immuno?uorescence staining was performed with antibodies against BMAL1 (green) and an astrocyte marker (GFAP, red) in brain sections. Nuclei were ?uorescently labeled with DAPI (blue). Arrow heads indicate BMAL1/GFAP-positive cells. (E) Double immuno?uorescence staining was performed with antibodies against BMAL1 (green) and a microglial marker (Iba1, red) in brain sections. Nuclei were ?uorescently labeled with DAPI (blue). Arrow heads indicate BMAL1/Iba1-positive cells. Representative images from the sham and ICH (24 h) groups are shown. (F) Relative fluorescence intensity of BMAL1 with NeuN (neurons), GFAP (astrocytes) and Iba1 (microglia) in the brain at 24 h after ICH. Scale bar =50 µm. All data are presented as the means ± SEM, **P<0.01 vs. Sham group; ***P<0.001 vs. Sham group; NS, no significant difference vs. Sham group. n=6. ICH, Intracerebral hemorrhage; DAPI, 4’,6-diamidino-2-phenylindole; SEM, standard error of mean.

Fig 3: The miR-155 expression level and level of the BMAL1 protein by inhibiting miR-155 after ICH. (A) The binding site of BMAL1 in the miR-155 3’UTR. (B) The levels of miR-155 were increased after ICH, and the increase was established at 3 h after ICH and remained at a high level. (C) Western blot analysis of the BMAL1 protein levels in the sham, ICH, ICH + antagomir-NC and ICH + antagomir-155 groups. (D) Quantification of the relative BMAL1 protein levels in the groups listed above. All data are presented as the means ± SEM, *P<0.05 vs. Sham group; **P<0.01 vs. Sham group; ***P<0.001 vs. Sham group; ##P<0.01 vs. ICH + antagomir-NC group; NS, no significant difference vs. ICH group; n=6. ICH, intracerebral hemorrhage; NC, negative control.

Fig 4: Effects of BMAL1 overexpression on oxidative stress, BBB injury, brain edema, inflammation and neurobehaviors after ICH. (A) Western blot analysis of levels of the BMAL1 and Albumin proteins (index of BBB injury) in the sham, ICH, ICH + Vector and ICH + Over-BMAL1 groups at 24 h after ICH. (B) Quantification of the relative BMAL1 protein levels in the four groups. (C) ROS levels in the brain tissues from rats in the four groups. (D) Quantification of the relative Albumin protein levels in the four groups. (E) The effects of BMAL1 overexpression on the brain water content of rats in the four groups. (F) Concentrations of IL-1ß in the serum of the sham ICH, ICH + Vector and ICH+Over-BMAL1 groups at 24 h after ICH. (G) Concentrations of TNF-a in the serum of the four groups listed above. (H) The scores on the modified Garcia test in the sham, ICH, ICH + Vector and ICH + Over-BMAL1 groups at 24 h after ICH. (I) Representative images illustrate swimming trajectories at 25 d (learning) and escape latency in the Morris water maze test at 21 to 25 d after ICH in the sham, ICH, ICH + Vector and ICH + Over BMAL1 rats. (J) Representative images illustrate swimming trajectories at 26 d (memory) and time spent in the platform quadrant in the Morris water maze test at 26 d after ICH in the groups listed above. **P<0.01 vs. Sham group; ***P<0.001 vs. Sham group; #P<0.05 vs. ICH + Vector group; ##P<0.01 vs. ICH + Vector group. NS, no significant difference vs. ICH group. In (A-G), n=6; In (H-J), n=10. BBB, blood brain barrier; ICH, intracerebral hemorrhage; ROS, reactive oxygen species.

Fig 5: Effects of BMAL1 overexpression on neuronal injury and degeneration induced by ICH. (A) Nissl staining was used to assess neuronal death in the sham, ICH, ICH + Vector and ICH+Over-BMAL1 groups at 24 h after ICH. Arrow heads indicate surviving neurons. Scale bar =50 µm. (B) Double immunofluorescence staining for NeuN (green) and TUNEL (red) was performed. Nuclei were fluorescently labeled with DAPI (blue) in the sham, ICH, ICH + Vector and ICH + Over-BMAL1 groups at 24 h after ICH. Arrows indicate dead neurons (TUNEL/NeuN-positive cells). Scale bar =50 µm. (C) FJB staining (green) was performed to assess neuronal degeneration in the groups listed above. Arrows indicate FJB-positive neurons. Scale bar =50 µm. (D) Quantitative analysis of surviving neurons in the cortex of each group. (E) Quantitative analysis of surviving neurons in the hippocampus region of each group. (F) Quantitative analysis of the percentages of TUNEL-positive dead neurons. (G) Quantitative analysis of FJB staining in brain sections from each group. FJB-positive cells were counted per 400× field. ***P<0.001 vs. Sham group; ##P<0.01 vs. ICH + Vector group; NS, no significant difference vs. ICH group. n=6. ICH, intracerebral hemorrhage; DAPI, 4’ 6-diamidino-2-phenylindole.