Additional file 1 of Fluoxetine inhibited the activation of A1 reactive astrocyte in a mouse model of major depressive disorder through astrocytic 5-HT2BR/β-arrestin2 pathway
Creators: name: Fang, Yinquan
name: Ding, Xiao
name: Zhang, Yihe
name: Cai, Lei
name: Ge, Yuan
name: Ma, Kaiyang
name: Xu, Rong
name: Li, Shanshan
name: Song, Mengmeng
name: Zhu, Hong
name: Liu, Jiaqi
name: Ding, Jianhua
name: Lu, Ming
name: Hu, Gang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. Primers and siRNA used in the study. Fig. S1. CMS-induced anxiety-like behaviors are reduced by fluoxetine administration. A Schematic diagram of CMS preferment and fluoxetine treatment experimental design. B-D The circuit diagram (B), bouts of mice in center (C) and duration of mice staying in center (D) in the OFT. E Latency time of mice to the first sniff in SIT. F Latency time of mice to the first feed &gt; 3 s in NSFT. Quantitative data are mean ± s.e. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. n = 8 per group, biologically independent animals. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Fig. S2. A1 astrocyte reactivity in cortex of mice is protected by fluoxetine administration. A Heatmap of A1-special and A2-special transcripts in the cortex as analyzed by RT-qPCR. ***P &lt; 0.001 vs control mice treated with saline; ###P &lt; 0.001 vs CMS mice treated with saline. B-C Expression of C3 in the cortex. D Protein levels of IL-6 in the serum, cortex and hippocampus. Quantitative data are mean ± s.e. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. n = 4 per group, biologically independent animals. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Fig. S3. Effects of fluoxetine on the releases of LDH in the supernatants of astrocyte. A Primary cultured microglia, astrocyte and neuron were sained with Iba 1, GFAP and Map2 (Scale bars, 50 µm), respectively. B–D Effects of fluoxetine on the releases of LDH in the supernatants of astrocyte after A1 (B), activated (LPS)-MCM (C) and IL-6 (D) stimulation. Quantitative data are mean ± s.e. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. All data represent the results of three independent experiments. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Fig. S4 Expression of 5-HT2BR in astrocyte after transfection with siRNA for 48h. A Representative blots. B Quantitative data shown in (A). Quantitative data are mean ± s.e. Data were analyzed using Student’s t test. All data represent the results of three independent experiments. ***P &lt; 0.001. Fig. S5 AAV-mediated depletion of 5-HT2BR in astrocyte. A Schematic representation of the AAV virus construct used to express 5-HT2BR or control siRNA in astrocyte under the gfaABC1D promoter. B Protein levels of 5-HT2BR in the hippocampus of mice after AAV injection for 4 weeks. Quantitative data are mean ± s.e. Data were analyzed using Student’s t test. All data represent the results of three independent experiments. ***P &lt; 0.001. C Expression of 5-HT2BR on astrocyte in the hippocampus DG of mice. Scale bars, 20 µm. Similar results were obtained in three separate experiments. D Schematic diagram of AAV injection experimental design. Fig. S6. Astrocytic 5-HT2BR knockdown reverses the functions of fluoxetine in anxiety-like behaviors and the expression of A1-special markers in cortex of mice. A–C The circuit diagram (A), bouts of mice in center (B) and duration of mice with or without fluoxetine treatment after AAV injection staying in center (C) in the OFT. D Latency time of mice to the first sniff in SIT. E Latency time of mice to the first feed &gt; 3 s in NSFT. F Levels of A1-special genes in the cortex of CON and CMS mice after AAV injection. Quantitative data are mean ± s.e. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. B-E: n = 8 (C, E), n = 9 (B, D) CON with Control AAV and saline; n = 8 (C, E), n = 9 (B, D) CON with Control AAV and FLX; n = 8 (D, E), n = 9 (B, C) CMS with Control AAV and saline; n = 8 (B, E), n = 9 (C, D) CMS with Control AAV and FLX; n = 8 CON with mHT2BR AAV and saline; n = 8 (D, E), n = 9 (B, C) CON with mHT2BR AAV and FLX; n = 8 (E), n = 9 (B–D) CMS with mHT2BR AAV and saline; n = 8 (B, D, E), n = 9 (C) CMS with mHT2BR AAV and FLX; F: n = 4 per group; biologically independent animals. *P &lt; 0.05, and ***P &lt; 0.001 vs respective control mice treated with saline; #P &lt; 0.05, and ###P &lt; 0.001 vs CMS mice injected with control AAV and treated with saline; $P &lt; 0.05, $$P &lt; 0.01, and $$$P &lt; 0.001. Fig. S7. β-arrestin 2 is contribute to the effects of fluoxetine on A1 astrocyte reactivity in vitro. A Expression of β-arrestin 1 in primary astrocyte isolated from WT and β-arrestin 1 knockout mice. B Expression of β-arrestin 2 in primary astrocyte of isolated from WT and β-arrestin 2 knockout mice. C Effects of β-arrestin 2 knockout on the levels of A1-special genes in astrocyte with or without fluoxetine treatment after activated (LPS)-MCM stimulation. Quantitative data are mean ± s.e. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. All data represent the results of three independent experiments. ***P &lt; 0.001 vs respective control cells treated with non-activated MCM; ##P &lt; 0.01, and ###P &lt; 0.001 vs WT control cells treated with LPS-MCM stimulation; $$P &lt; 0.01, and $$$P &lt; 0.001. Fig. S8. β-arrestin 2 knockout reduces the effects of fluoxetine on the expression of A1-special markers in cortex of CMS mice. A-C The circuit diagram (A), bouts of WT and β-arrestin 2 knockout mice in center (B) and duration of WT and β-arrestin 2 knockout mice staying in center (C) in the OFT. D Latency time of mice to the first sniff in SIT. E Latency time of mice to the first feed &gt; 3 s in NSFT. F Levels of A1-special genes in the cortex of WT and β-arrestin 2−/− mice. Data were analyzed using two-way ANOVA, then combined with Tukey’s multiple comparison to assess the differences between groups. Quantitative data are mean ± s.e. B-E: n = 8 (D, E), n = 9 (B, C) WT CON with saline; n = 8 (C-E), n = 9 (B) WT CON with FLX; n = 8 (D, E), n = 10 (B, C) WT CMS with saline; n = 8 (D, E), n = 9 (B, C) WT CMS with FLX; n = 8 (E), n = 9 (B, C), n = 10 (D) β-arrestin 2−/− CON with saline; n = 8 (B, D, E), n = 9 (C) β-arrestin 2−/− CON with FLX; n = 8 β-arrestin 2−/− CMS with saline; n = 8 (D, E), n = 9 (B, C) β-arrestin 2−/− CMS with FLX; F: n = 4 per group; biologically independent animals. ***P &lt; 0.001 vs respective control mice treated with saline; #P &lt; 0.05, and ###P &lt; 0.001 vs WT CMS mice treated with saline; $P &lt; 0.05, $$P &lt; 0.01, and $$$P &lt; 0.001. Fig. S9. The overall experimental design of this study.