Journal: CNS neuroscience & therapeutics

Article Title: NOTCH3 Mutation Causes Glymphatic Impairment and Promotes Brain Senescence in CADASIL.

doi: 10.1111/cns.70140

Figure Lengend Snippet: FIGURE 4 | Activation of RUNX1-CMYB-AQP4 signaling is downregulated in NOTCH3-R170C astrocyte. (A) Dual-luciferase reporter assays showing the effect of CMYB activation on Aqp4 transcription in HEK293T cells. Experiments were repeated for three times. *p < 0.05; by Student's t test (mean ± SEM). (B) Total or nucleus protein of NOTCH3-R170C and -WT astrocytes were subjected to western blot. Protein level was normalized to BACT or LAMIN B1 expression. Experiments were repeated for four times. *p < 0.05, **p < 0.01, and ***p < 0.001; by Student's t test (mean ± SEM). (C) Protein was extracted from NOTCH3-R170C or -WT astrocyte then subjected to immunoprecipitation (IP) with anti-NOTCH3 or anti-CMYB antibodies. IP products were analyzed with western blot to examine the interaction of NOTCH3 and RUNX1 or RUNX1 and CMYB, respectively. Experiments were repeated for 3 times. *p < 0.05; by Student's t test (mean ± SEM). (D) Immunostaining of CMYB (green) and RUNX1 (red) with NOTCH3-R170C or -WT astrocyte. (E, F) NOTCH3-R170C or -WT OSCs were labeled with RUNX1 (green) and S100B (red) (E) or CMYB (green) and S100B (red) (F), respectively. Percentage of RUNX1+ or CMYB+ cells among S100B+ astrocytes was calculated. Experiments were repeated for three times. *p < 0.05, **p < 0.01; by Student's t test (mean ± SEM). (G–H) Coronal brain sections of NOTCH3-R170C or -WT mice (16 weeks of age) were subjected to immunostaining of RUNX1 (red) and S100B (green) (G) or CMYB (red) and S100B (green) (H). N = 6 in each group. *p < 0.05, **p < 0.01; by Student's t test (mean ± SEM). (I) RUNX1 was overexpressed in NOTCH3-R170C or -WT astrocytes with lentiviral vectors carrying Runx1 cDNA (Runx1-OE) which were then subjected to western blot. Protein level was normalized to GAPDH expression. Experiments were repeated for four times. *p < 0.05, **p < 0.01; by one-way ANOVA (mean ± SEM).

Article Snippet: The following primary antibodies were used: rabbit anti- AQP4 (Proteintech 16,473- 1- AP, 1:1000), rabbit anti- RUNX1 (Affinity Biosciences AF6379, 1:1000), rabbit antiCMYB (Proteintech 17,800- 1- AP, 1:1000), rabbit anti- NOTCH3 (Proteintech 55,114- 1- AP, 1:1000), mouse anti- GAPDH (Proteintech 60,004- 1- Ig, 1:10000), mouse anti- β- ACTIN (Proteintech 66,009- 1- Ig, 1:10000), and mouse anti- LAMIN B1 (Proteintech 66,095- 1- Ig, 1:10000).

Techniques: Activation Assay, Luciferase, Western Blot, Expressing, Immunoprecipitation, Immunostaining, Labeling