Additional file 1 of Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity
Creators: name: Kong, Shuo
name: Chen, Tao-xiang
name: Jia, Xiang-lei
name: Cheng, Xue-lei
name: Zeng, Meng-liu
name: Liang, Jing-yi
name: He, Xiao-hua
name: Yin, Jun
name: Han, Song
name: Liu, Wan-hong
name: Fan, Yuan-teng
name: Zhou, Ting
name: Liu, Yu-min
name: Peng, Bi-wen
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Marine Biology
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. The interference efficiency measurement of si.NFIA in mice. (A) Schema of si.NC/ NFIA delivery into the hippocampus region in mice. Representative immunoblot bands of NFIA (Bi) and its band density analysis (Bii) (n = 3, *** P &lt; 0.001, One-way ANOVA). Figure S2. Effects of intraperitoneal 4-AP on NFIA expression in mouse hippocampal neurons and microglia. A Representative confocal images showing NFIA+ neurons and B NFIA+ microglia in hippocampal CA1, CA2, CA3 and DG from the mice with ip.4-AP or saline. (n = 6 for each group, Student’s t test). Figure S3. Effects of NFIA deficiency on the conversion of the astrocytic phenotype induced by 4-AP. A Scheme of primary astrocyte preparation for immunofluorescent staining and protein analysis. B Representative confocal images (Bi) of GFAP and C3 and the fluorescence intensity analysis of (Bii) GFAP and (Biii) C3 in primary cultured astrocytes transfected with si.NC or si.NFIA in the presence or absence of 4-AP (n = 6 for each group, ***p &lt; 0.001, One-way ANOVA). Representative immunoblot bands (Ci) of S100A10, GFAP, NFIA and C3, and the band density analysis of NFIA (Cii), GFAP (Ciii), S100A10 (Civ) and C3 (Cv) in primary cultured astrocytes transfected with si.NC or si.NFIA in 4-AP exposure or not (n = 3 for each group, **P &lt; 0.01, ***P &lt; 0.001, One-way ANOVA). Figure S4. Effects of 4-AP on the expression of NFIA and TRPV4 in mouse hippocampal tissues and in primary cultured astrocytes. A The qPCR analysis showing mRNA levels of NFIA (Ai) and TRPV4 (Aii) in the hippocampus from the mice with intraperitoneal 4-AP or saline injection (n = 5 for each group. * P &lt; 0.05, ** P &lt; 0.01, Student’s t test). Representative immunoblot bands (Bi) and the band intensity analysis exhibiting protein levels of GFAP (Bii), NFIA (Biii) and TRPV4 (Biv) in hippocampal tissues from the mice with intraperitoneal 4-AP or saline injection (n = 3 for each group, *** P &lt; 0.001, Student’s t test). Representative confocal immunofluorescent images of NFIA (Ci) and TRPV4 (Cii) and their immunofluorescence intensity analysis in primary cultured astrocytes with or without 4-AP treatment (n = 6 for each group, ***P &lt; 0.001, Student’s t test). The qPCR analysis of NFIA (Di) and TRPV4 (Dii) mRNA levels in primary cultured astrocytes with or without 4-AP treatment (n = 4 for each group, *P &lt; 0.05, **P &lt; 0.01, Student’s t test). Representative immunoblot bands (Ei) of NFIA and TRPV4 in primary cultural astrocytes with or without 4-AP treatment and the band density analysis of NFIA (Eii), GFAP (Eiii) and TRPV4 (Eiv) (n = 3 for each group, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, Student’s t test). Figure S5. Effects of hippocampal si.NFIA treatment on the expression of TRPV4. A Representative immunoblot bands of NFIA and TRPV4 in hippocampal tissues from the mice treated with si.NC or si.NFIA and their band density analysis (B) (n = 3 for each group, ***P &lt; 0.001, Student’s t test). Figure S6. Effects of astrocytic NFIA knockdown on TRPV4 upregulation and cytosolic calcium level. A Representative immunoblot bands of NFIA and TRPV4, and the band density analysis of NFIA and TRPV4 in astrocytes transfected with si.NC or si.NFIA in the presence or absence of 4-AP (n = 3 for each group, **P &lt; 0.001, ***P &lt; 0.001, ****P &lt; 0.0001, One-way ANOVA). Bi Representative confocal images of the Ca2+ indicator Fluo 4 and the fluorescence intensity analysis (Bii) showing the basal Ca2+ level in primary cultured astrocytes transfected with si.NC or si.NFIA in the presence or absence of 4-AP (n = 8 for each group, *p &lt; 0.05, ****p &lt; 0.0001, One-way ANOVA). Figure S7. Hippocampal astrocytic NFIA knockdown via rAAV infection targeting astrocyte. A Scheme of experiments on hippocampal rAAV delivery and immunofluorescent staining. B Representative confocal images of immunofluorescent staining showing NFIA expression in rAAV-GfaABC1D-sh.NC-infected group versus rAAV-GfaABC1D-sh. NFIA-infected group.