Additional file 1 of Captive ERVWE1 triggers impairment of 5-HT neuronal plasticity in the first-episode schizophrenia by post-transcriptional activation of HTR1B in ALKBH5-m6A dependent epigenetic mechanisms
Creators: name: Wu, Xiulin
name: Liu, Lianzhong
name: Xue, Xing
name: Li, Xuhang
name: Zhao, Kexin
name: Zhang, Jiahang
name: Li, Wenshi
name: Yao, Wei
name: Ding, Shuang
name: Jia, Chen
name: Zhu, Fan
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology
subject: Mental Health

description: Additional file 1: Table S1. The concentration of SERT in the plasma of healthy controls and schizophrenia patients. Table S2. The concentration of TPH2 in the plasma of healthy controls and schizophrenia patients. Table S3. The concentration of 5-HT in the plasma of healthy controls and schizophrenia patients. Table S4. The concentration of ERVWE1 in the plasma of healthy controls and schizophrenia patients. Table S5. Comparison of the whole peripheral blood samples demographic data between the healthy controls and recent-onset schizophrenia patients. Table S6. Comparison of the plasma samples demographic data between the healthy controls and recent-onset schizophrenia patients. Table S7. Primer sequences used in real-time quantitative PCR (RT-qPCR). Table S8. Primer sequences used in plasmid constructs and siRNA. Table S9. Antibodies used in western blot. Fig S1. 5-HT receptors abnormality in the prefrontal cortex (BA46) of schizophrenia by GSE53987. A-F Boxplot of 5-HT receptors expression in schizophrenia (n = 15) vs healthy controls (n = 19), A Boxplot of HTR2A expression, B Boxplot of HTR1A expression, C Boxplot of HTR2B expression, D Boxplot of HTR5A expression, E Boxplot of HTR6 expression, F Boxplot of HTR7 expression. p-valve was analyzed by wilcoxon (Mann-Withney). Data shown are the mean ± SD. Fig S2. mRNA expression level of 5-HTergic systems, Arc, and ALKBH5 in the whole peripheral blood of schizophrenia patients and healthy controls. A-I Respectively represent the mRNA expression levels of HTR1B, HTR1A, HTR2A, HTR6, HTR7, SERT, TPH2, Arc, and ALKBH5 in the whole peripheral blood of schizophrenia patients (n = 15) and healthy controls (n = 14) by RT-qPCR (p-value by median and nonparametric analysis). J-L Correlation of ERVWE1 mRNA level with HTR1B (p = 0.01, r = 0.65), Arc (p = 0.03, r = 0.57), and ALKBH5 (p &lt; 0.01, r = 0.78) mRNA levels in schizophrenia by Spearman. Dots depict schizophrenia patients, but a few are overlapping and cannot be separated on the graph. Data shown are the mean ± SD. Fig S3. ERVWE1 overexpressed in the SH-SY5Ycells. A The representative western blot results of ERVWE1. B Protein expression level of ERVWE1. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: Student’s t-test (*p &lt; 0.05). Fig S4. HTR1B activated ERK-ELK1-Arc signal pathway in the SH-SY5Y cells. A-D Representative western blot results for ERK1/2, p-ERK1/2, ELK1, p-ELK1, and Arc proteins in the SH-SY5Y cells after transfection with HTR1B expressed vectors and siRNA oligo respectively. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: Student’s t-test (ns p &gt; 0.05, *p &lt; 0.05, **p &lt; 0.01). Fig S5. ERVWE1 activated HTR1B signal pathway in SH-SY5Y cells. The protein expression levels of ERVWE1 and HTR1B were detected in cells co-transfected with ERVWE1 and siHTR1B. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: one-way ANOVA (*p &lt; 0.05, **p &lt; 0.01). Fig S6. ERVWE1 activated HTR1B signal pathway in DRN neurons. A Transfection efficiency of ERVWE1 in DRN neurons by western blot. B Immunofluorescence staining with HTR1B in DRN neurons. C The western blot results of ERVWE1 and HTR1B were detected by applying siHTR1B and ERVWE1. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: Student’s t-test and one-way ANOVA (*p &lt; 0.05, **p &lt; 0.01). Fig S7. The transfection efficiency of ERVWE1 in the DRN neurons. Scale bars = 25 μm. Fig S8. ALKBH5 regulated m6A modification in SHSY5Y. A, B RNA dot blot analysis of m6A levels. A ALKBH5 expressed cells vs control cells, B siALKBH5 cells and siNC cells. Methylene blue staining served as a loading control. C-E Western blot results of ALKBH5 and HTR1B. C, D ALKBH5 overexpression, E ALKBH5 knockdown. F The western blot results of ERVWE1 and ALKBH5 were detected when ERVWE1 co-transfected with siALKBH5 or siNC control. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: Student’s t-test and one-way ANOVA (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001). Fig S9. m6A modification sites predictor of HTR1B mRNA. A SRAMP. High and very high confidences were considered as a candidate site. B RMBase v2.0. Score above 400 was considered as a candidate site. Fig S10. ERVWE1 reduced the m6A level of HTR1B by 198 and 574 sites. A Schematic illustration of HTR1B with either wild-type or mutant m6A sites luciferase activity plasmids constructs. B Relative luciferase activity of HTR1B-CDS wild-type and mutant m6A sites after co-transfection with ALKBH5 expressing plasmid or control plasmid in SH-SY5Y cells. C Decay of HTR1B mRNA with ERVWE1 overexpression versus control in the HTR1B-CDS-Mut1212 groups in SH-SY5Y cells. D The relative protein levels of ERVWE1 in SH-SY5Y cells. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: Student’s t-test (ns p &gt; 0.05, *p &lt; 0.05, ***p &lt; 0.001). Fig S11. ALKBH5 participated in ERVWE1-induced Arc upregulation in DRN neurons. A Immunofluorescence staining with Arc in DRN neurons. B The relative intensity of Arc. Data shown are the mean ± SD and represent three independent experiments. Statistical analysis: one-way ANOVA (**p &lt; 0.01).