Additional file 1 of The Schwann cell-specific G-protein Gαo (Gnao1) is a cell-intrinsic controller contributing to the regulation of myelination in peripheral nerve system
Creators: name: Xu, Jinghui
name: Peng, Qianqian
name: Cai, Jieyi
name: Shangguan, Jianghong
name: Su, Wenfeng
name: Chen, Gang
name: Sun, Hualin
name: Zhu, Changlai
name: Gu, Yun
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Computational Biology

description: Additional file 1: Fig. S1. Gnao1 expression in nerve tissue (sciatic nerves and spinal cords) and myelination-related cells (neurons and SCs) by RT-qPCR (A) and WB analysis (B). Fig. S2 Validation of Gnao1-shRNAs interference efficiency. A RT-qPCR comparing the mRNA levels of Gnao1 in SCs treated with Gnao1-shRNAs or Scramble (NC-shRNA) for 48 h. T-test, n = 3, **, p &lt; 0.01 and ***, p &lt; 0.001 vs NC-shRNA. B WB comparing the protein levels of Gnao1 in SCs treated with Gnao1-shRNAs or Scramble for 48 h. T-test, n = 3, *, p &lt; 0.05 and **, p &lt; 0.01 vs NC-shRNA. Fig. S3 Knockdown or overexpression of Gnao1 level in Schwann cells of mouse sciatic nerve by injection of virus carried with Gnao1-shRNA or mouse Gnao1 coding sequence, respectively. A Schematic diagram illustrates the experimental process, that is, by respectively injecting the viruses carrying the Gnao1-shRNA or coding sequences into sciatic nerve, to generate mice with Gnao1 knockdown or overexpression in the sciatic nerve (referred to as Gnao1-NKD and Gnao1-NOE-mice). B and C WB comparing the protein levels of Gnao1 in Gnao1-NKD-mice (B), Gnao1-NOE-mice (C) together with their own controls, showing the lower Gnao1 expression in Gnao1-NKD-mice and higher Gnao1 expression in Gnao1-NOE-mice compared to controls, and IHC showing the Gnao1 knockdown or overexpression occurred mainly in the SCs of the sciatic nerves. Scale bar = 100 μm. T-test, n = 3, **, p &lt; 0.01 and ****, p &lt; 0.0001 vs controls. Fig. S4 Knockdown Gnao1 expression in neurons of mouse spinal cord by injection of virus carried with Gnao1-shRNA. A Schematic diagram illustrates the experimental process, that is, by intrathecally injecting the viruses carrying the Gnao1-shRNA into spinal cord, to generate mice with Gnao1 knockdown in the spinal cord (referred to as Gnao1-SKD-mice). B WB comparing the protein levels of Gnao1 in Gnao1-SKD-mice together with controls, showing the lower Gnao1 expression in Gnao1-SKD-mice compared to controls. T-test, n = 3, **, p &lt; 0.01 vs controls. C IHC showing the Gnao1 knockdown occurred mainly in the spinal cord neurons. Scale bar = 500 μm. Zoomed in is the enlargement of the white box area, scale bar = 200 μm. Fig. S5. Analysis of RNA sequencing data of Gnao1-KD-SCs and control (NC) before and after differentiation. A Bar chart showing the number of differentially expressed genes (DEGs) obtained by pair-to-pair comparison of NC-SCs, Gnao1-KD-SCs, Diff-NC-SCs and Diff-Gnao1-KD-SCs (cutoff: fold change (FC) &gt; 1.5 or &lt; 0.67 plus p-value &lt; 0.05). Bb1-Ee1 Heat map showing representative DEGs in 4 datasets (i.e., 1960 DEGs in Gnao1-KD-SCs versus NCs (b1), 3607 DEGs in Diff-NC-SCs versus NCs (c1), 4672 DEGs in Diff-Gnao1-KD-SCs versus Gnao1-KD-SCs (d1), and 161 DEGs in Diff-Gnao1-KD-SCs versus Diff-NC-SCs (e1)) by cluster heatmap analysis. Bb2-Ee2 Bubble map showing the top 20 functions of upregulated DEGs in 4 datasets (i.e., Gnao1-KD-SCs versus NCs (b2), Diff-NC-SCs versus NCs (c2), Diff-Gnao1-KD-SCs versus Gnao1-KD-SCs (d2), and Diff-Gnao1-KD-SCs versus Diff-NC-SCs (e2)) by KEGG enrichment analysis. Fig. S6. Analyze of myelination-related genes in RNA sequencing data with heat map cluster and qPCR. A Heat map showing representative myelination-related DEGs in 4 datasets (i.e., DEGs in Gnao1-KD-SCs versus NCs, Diff-NC-SCs versus NCs, Diff-Gnao1-KD-SCs versus Gnao1-KD-SCs, and Diff-Gnao1-KD-SCs versus Diff-NC-SCs) by cluster heatmap analysis. B Histogram showing the relative expression of (b1) myelin-forming proteins (MPZ, MBP, PMP22 and MAG), (b2) positive transcriptional regulators of myelination (EGR2, Pou3F1, Pou3F2 and Id2), (b3) the molecules that have been proven to affect myelination (Dok4, Vamp7, Erbb2, Erbb3, Cadm3 and Adcy2), and others (Gnao1 and Adcy2) in NC-SCs, Gnao1-KD-SCs, Diff-NC-SCs and Diff-Gnao1-KD-SCs. Pair t-test, n = 3, Ns, p &gt; 0.05, no statistical difference. *, p &lt; 0.05, **, p &lt; 0.01, and ***, p &lt; 0.001