Journal: Cell reports

Article Title: Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia.

doi: 10.1016/j.celrep.2023.112869

Figure Lengend Snippet: Figure 4. PI3K/AKT signaling pathway mediates Sox10 phosphorylation (A) GSEA of scRNA-seq data showing the enrichment of phosphatidylinositol signaling system and MAPK signaling pathway in the contractile VSMCs (clusters 2) and macrophage-like VSMCs (cluster 0) of the neointima. NES, normalized enrichment score. (B) KEGG pathway enrichment of DEGs between the contractile VSMCs (clusters 2) and macrophage-like VSMCs (cluster 0) of the neointima in scRNA-seq analysis. (C) Representative western blot analysis of protein expression of PI3K, p-PI3K, AKT, and p-AKT in mouse VSMCs treated with TNF-a for 5 min or 24 h. (D) Immunoprecipitation and representative western blot analysis for P-ser of Sox10 in VSMCs pretreated with or without PI3K inhibitor ZSTK474, AKT activator SC79, and AKT inhibitor (E)-AKTIV followed by TNF-a treatment. (E) Representative western blot analysis of protein expression of MEK, p-MEK, JNK, p-JNK, p38, p-p38, ERK, and p-ERK in VSMCs treated with TNF-a. (F) Immunoprecipitation and representative western blot analysis for P-ser of Sox10 in VSMCs pretreated with or without ERK inhibitor FR180204 followed by TNF-a treatment. (G) Representative western blot analysis of protein expression of ERK and p-ERK in VSMCs pretreated with or without AKT inhibitor (E)-AKTIV followed by TNF-a treatment.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER HRP goat anti-rabbit Proteintech Cat#SA00001-2; RRID: AB_2722564 HRP goat anti-mouse Proteintech Cat# SA00001-1; RRID: AB_2722565 Biological samples Human aortic samples Department of Cardiac Surgery, the Fourth Hospital of Hebei Medical University This manuscript Human renal artery samples Department of Cardiac Surgery, the Fourth Hospital of Hebei Medical University This manuscript Chemicals, peptides, and recombinant proteins TNF-a R&D Cat# 210-TA-100 PI3K inhibitor ZSTK474 MCE Cat# HY-50847 AKT activator SC79 MCE Cat# HY-18749 AKT inhibitor (E)-AKTIV MCE Cat# HY-14971 ERK inhibitor FR180204 MCE Cat# HY-12275 HDAC inhibitor trichostatin A (TSA) Selleck Cat# S1045 HDAC activator ITSA-1 MCE Cat# HY-100508 Gaq/11 inhibitor pertussis toxin (PTX) Sigma Cat# 70323-44-3 Critical commercial assays Lactate Assay Kit Elabscience Cat# E-BC-K044-M Chemiluminescent EMSA Kit Beyotime Cat# GS009 DuolinkTM In Situ PLA Probe anti-Rabbit PLUS Sigma-Aldrich Cat# DUO92002-30RXN DuolinkTM In Situ PLA Probe anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004-30RXN DuolinkTM In Situ Detection Reagents Orange Sigma-Aldrich Cat# DUO92007-30RXN BeyoMagTM Anti-GFP Magnetic Beads Beyotime Cat# P2132-2mL BeyoMagTM Anti-HA Magnetic Beads Beyotime Cat# P2121-2mL BeyoMagTM Anti-GST Magnetic Beads Beyotime Cat# P2138-2mL Hyperactive pG-MNase CUT&RUN Kit Vazyme Cat# HD102 VAHTS Multiplex Oligos Set 4 for Illumina Kit Vazyme Cat# N321 Dual-Luciferase Reporter Assay System Promega Cat# E1910 Deposited data scRNA-seq data Gao et al.22 GEO:GSE194173 Experimental models: Cell lines Human embryonic kidney (HEK293A) ATCC Cat# CRL-1573 RAW264.7 ATCC Cat# TIB-71 Experimental models: Organisms/strains Myh11-CreERT2-mTmG reporter mice Youdu Bio YDS0816/YDS0815 RGS5-KO mice Institute of Model Animals, Wuhan University N/A C57BL/6J mice Charles River N/A ApoE / mice Charles River N/A Oligonucleotides Primers for qPCR, see Table S1 This manuscript N/A siRNA sequences, see Table S2 This manuscript N/A PCR primers for ChIP assay, see Table S3 This manuscript N/A Oligonucleotide sequences for EMSA assays, see Table S4 This manuscript N/A Recombinant DNA pEZX-FR01 plasmid General Biosystems N/A pcDNA3.1 plasmid General Biosystems N/A (Continued on next page) Cell Reports 42, 112869, August 29, 2023 15

Techniques: Phospho-proteomics, Western Blot, Expressing, Immunoprecipitation

Journal: Cell reports

Article Title: Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia.

doi: 10.1016/j.celrep.2023.112869

Figure Lengend Snippet: Figure 5. Sox10 phosphorylation is suppressed by RGS5 via blocking PI3K/AKT signaling (A) Transcriptomic analysis of DEGs in ligation and healthy carotid artery in WT mice (n = 30). (B) UMAP plot showing all VSMCs in mouse healthy carotid artery colored by clusters. (C) Expression level of RGS5 in each cluster in the healthy carotid artery of WT mice by scRNA-seq analysis. (D) Immunoprecipitation and representative western blot analysis for P-ser of Sox10 in VSMCs transfected with si-Con or si-RGS5 followed by TNF-a treatment.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER HRP goat anti-rabbit Proteintech Cat#SA00001-2; RRID: AB_2722564 HRP goat anti-mouse Proteintech Cat# SA00001-1; RRID: AB_2722565 Biological samples Human aortic samples Department of Cardiac Surgery, the Fourth Hospital of Hebei Medical University This manuscript Human renal artery samples Department of Cardiac Surgery, the Fourth Hospital of Hebei Medical University This manuscript Chemicals, peptides, and recombinant proteins TNF-a R&D Cat# 210-TA-100 PI3K inhibitor ZSTK474 MCE Cat# HY-50847 AKT activator SC79 MCE Cat# HY-18749 AKT inhibitor (E)-AKTIV MCE Cat# HY-14971 ERK inhibitor FR180204 MCE Cat# HY-12275 HDAC inhibitor trichostatin A (TSA) Selleck Cat# S1045 HDAC activator ITSA-1 MCE Cat# HY-100508 Gaq/11 inhibitor pertussis toxin (PTX) Sigma Cat# 70323-44-3 Critical commercial assays Lactate Assay Kit Elabscience Cat# E-BC-K044-M Chemiluminescent EMSA Kit Beyotime Cat# GS009 DuolinkTM In Situ PLA Probe anti-Rabbit PLUS Sigma-Aldrich Cat# DUO92002-30RXN DuolinkTM In Situ PLA Probe anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004-30RXN DuolinkTM In Situ Detection Reagents Orange Sigma-Aldrich Cat# DUO92007-30RXN BeyoMagTM Anti-GFP Magnetic Beads Beyotime Cat# P2132-2mL BeyoMagTM Anti-HA Magnetic Beads Beyotime Cat# P2121-2mL BeyoMagTM Anti-GST Magnetic Beads Beyotime Cat# P2138-2mL Hyperactive pG-MNase CUT&RUN Kit Vazyme Cat# HD102 VAHTS Multiplex Oligos Set 4 for Illumina Kit Vazyme Cat# N321 Dual-Luciferase Reporter Assay System Promega Cat# E1910 Deposited data scRNA-seq data Gao et al.22 GEO:GSE194173 Experimental models: Cell lines Human embryonic kidney (HEK293A) ATCC Cat# CRL-1573 RAW264.7 ATCC Cat# TIB-71 Experimental models: Organisms/strains Myh11-CreERT2-mTmG reporter mice Youdu Bio YDS0816/YDS0815 RGS5-KO mice Institute of Model Animals, Wuhan University N/A C57BL/6J mice Charles River N/A ApoE / mice Charles River N/A Oligonucleotides Primers for qPCR, see Table S1 This manuscript N/A siRNA sequences, see Table S2 This manuscript N/A PCR primers for ChIP assay, see Table S3 This manuscript N/A Oligonucleotide sequences for EMSA assays, see Table S4 This manuscript N/A Recombinant DNA pEZX-FR01 plasmid General Biosystems N/A pcDNA3.1 plasmid General Biosystems N/A (Continued on next page) Cell Reports 42, 112869, August 29, 2023 15

Techniques: Phospho-proteomics, Blocking Assay, Ligation, Expressing, Immunoprecipitation, Western Blot, Transfection