Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 1. PRRC2B deficiency in oligodendroglia cells leads to developmental hypomyelination (A) Immunostaining of PRRC2B/PDGFRa/OLIG2 in the corpus callosum (CC) of mice with indicated genotypes. Scale bars, 25 mm. (B) Left: western blotting analysis of the protein levels of PRRC2B in the cerebral cortex from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P6. Right: quantification of PRRC2B protein levels. Values are shown as means ± SEMs, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 mice). (C) Quantitative real-time PCR detected the mRNA levels of Prrc2b in the cerebral cortex of P6 mice. Values are means ± SEMs. ***p < 0.001, unpaired two-tailed Student’s t test (each group, n = 9 replicates from >3 mice). (D) Pictures of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice and brains at P15. Scale bars, 5 mm (top), 2 cm (bottom).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Immunostaining, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 2. PRRC2B promotes OL progenitor cell differentiation (A) Immunofluorescence of PDGFRa/OLIG2 in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (B) Quantification of PDGFRa/OLIG2-positive OPCs in the CC of mice, with the indicated geno- types at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, NS, not statistically significant, unpaired two-tailed Student’s t test (P15: f/f, n = 19, f/f; Olig2, n = 13; P40: f/f, n = 14, f/f; Olig2, n = 6). (C) Immunostainings of CC1/OLIG2 in the CC of Prrc2bf/f, and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (D) Quantification of CC1/OLIG2-positive OLs in the CC of mice, with the indicated genotypes at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test (P15: f/f, n = 18, f/f; Olig2, n = 12; P40: f/f, n = 7, f/f; Olig2, n = 6). (E) Left: western blotting analysis of OPC devel- opment-related protein in the CC of mice at P15. Right: quantification of protein levels of OLIG2, MAG, MBP, and ALDH1L1. Values are shown as means ± SEMs, *p < 0.05, NS, not statistically significant, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OPC-related genes in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15. Values were shown as means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Stu- dent’s t test (n R 4 independent biological repli- cates for each genotype).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 3. PRRC2B deficiency affects OL progenitor cell differentiation (A) Immunostainings of PRRC2B/PDGFRa/OLIG2 in OPCs isolated from brain tissues of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4. Scale bars, 50 mm. (B) Western blotting analysis of the protein levels of PRRC2B in OPC-derived cells cultured in prolifera- tion medium for 4 days and quantification of PRRC2B protein levels. Values are shown as means ± SEMs, **p < 0.01, unpaired two-tailed Student’s t test (n = 4 mice). (C) Quantitative real-time PCR analysis of the mRNA levels of Prrc2b in OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs, ***p < 0.001, unpaired two-tailed Student’s t test (n = 6 independent biological replicates for each group). (D) Western blotting analysis the protein levels of OLIG2, ALDH1L1, and MBP in OPC-derived cells cultured in differentiation medium for 4 days. (E) Quantification of OLIG2, MBP, and ALDH1L1 protein levels. Values are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OL-related genes in OPCs-derived cells isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Isolation, Western Blot, Derivative Assay, Cell Culture, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 4. PRRC2B is an m6A-specific bind- ing protein in the brain (A) Schematic diagram of RNA pull-down showing that the unmethylated oligo(A) and methylated oligo-m6A RNA were used to capture potential m6A binding proteins in vitro. (B) Western blotting displaying endogenous PRRC2B and YTHDF1 pulled down in the brains of P4 mice. (C) LC-MS/MS quantification of the m6A/A ratio in RNA isolated from OPCs of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs. Un- paired two-tailed Student’s t test (n = 4 experi- ments for each group). (D) Left: HA-tag was added to the C terminus of Prrc2b in mice. Right: western blotting of the effi- ciency of HA-tag expression in whole brains of WT and Prrc2b-HA mice at P4. (E) Schematic diagram of the RIP assay for LC-MS/ MS and sequencing. (F) Quantification of the m6A/A ratio in mRNA iso- lated from the cerebral cortex of WT and Prrc2b- HA mice at P4 by RIP. Values are means ± SEMs of 3 independent experiments, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 independent biological replicates). (G) Pie chart presenting the distribution of PRRC2B-binding peaks in the 30 UTR, 50 UTR, CDS, or noncoding regions. (H) Enrichment chart depicting the proportion of PRRC2B-binding peaks in the corresponding 4 regions. (I) Binding motif identified by HOMER with PRRC2B-binding peaks (p = 1e12).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Methylation, Binding Assay, In Vitro, Western Blot, Liquid Chromatography with Mass Spectroscopy, Isolation, Two Tailed Test, Expressing, Sequencing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 5. PRRC2B regulates OL develop- ment in an m6A modification-dependent manner (A) Distribution of m6A peaks across the length of the mRNA (50 UTR, CDS, and 30 UTR). (B) Enriched motif identified by HOMER with m6A peaks in OPCs isolated from C57BL/6J mice at P4 (p = 1e122). (C) GO terms in the biological process category enriched in transcripts with downregulated expression levels in OPCs isolated from Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Expressing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 6. Sox2 is the target gene of PRRC2B during OPC development (A) Primary OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4 were exposed to ActD, and RNA was extracted at the indicated time points. Quantitative real-time PCR was per- formed to assess the half-life of Sox2 mRNA. The data are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (T1/2(f/f) = 44.99 min; T1/2(f/f; Olig2) = 20.02 min). (B) Integrative Genomics Viewer (IGV) tracks showing the read distributions of RNA-seq (top, n = 3), PRRC2B RIP-seq (center, n = 2), and MeRIP-seq (bottom, n = 3) profiling of Sox2 gene, with significant peaks highlighted in yel- low. Dark green indicates normalized RNA-seq profiles of OPCs isolated from Prrc2b cKO mice at P4, and gray shows that of OPCs of Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Real-time Polymerase Chain Reaction, Two Tailed Test, RNA Sequencing

Journal: British Journal of Cancer

Article Title: Immune evasion in lung metastasis of leiomyosarcoma: upregulation of EPCAM inhibits CD8 + T cell infiltration

doi: 10.1038/s41416-024-02576-z

Figure Lengend Snippet: a , b TYLMS-1 cells treated with EPCAM inhibitors-produced conditioned media. Tumour necrosis factor-α converting enzyme (TACE) inhibitor (TAPI), and γ-secretase inhibitor (DAPT) were used. In addition to the inhibition of EPCAM signalling by the combination of TAPI and DAPT, partial inhibition of EPCAM signalling was performed with each drug alone. The migration of CD8 + T cells was evaluated and compared using a Transwell assay with conditioned medium. c – f TYLMS-1 cells were transfected with small interfering (si)EPCAM or scrambled siRNA-produced conditioned medium. The migration of CD8 + T cells was evaluated and compared using a Transwell assay with conditioned medium. a , e The membrane of the Transwell insert was stained, and the number of migrated CD8 + T cells was evaluated. Representative microscopy images are shown. b , f Comparison of the numbers of migrated CD8 + T cells. For each membrane shown in ( a ) and ( e ), the number of stained positive cells was counted in five different fields using a 10× objective lens, and the total number was evaluated. c EPCAM expression in each cell line was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). d EPCAM expression in each cell line was determined by western blotting. Data in ( b ) and ( f ) are mean ± standard deviation (SD); n = 3. Two-tailed t -test. * p < 0.05, ** p < 0.01.

Article Snippet: TYLMS-1 and TC616 cells were treated to inhibit epithelial cellular adhesion molecule (EPCAM) signalling with 40 μM tumour necrosis factor-α converting enzyme (TACE) inhibitor (TAPI)-1 (Selleck Chemicals, Houston, TX, USA), 10 μM γ-secretase inhibitor (DAPT) (Selleck Chemicals), or a combination of both inhibitors when the cells reached ~60% confluence.

Techniques: Produced, Inhibition, Migration, Transwell Assay, Transfection, Membrane, Staining, Microscopy, Comparison, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Standard Deviation, Two Tailed Test

Journal: British Journal of Cancer

Article Title: Immune evasion in lung metastasis of leiomyosarcoma: upregulation of EPCAM inhibits CD8 + T cell infiltration

doi: 10.1038/s41416-024-02576-z

Figure Lengend Snippet: a Volcano plot showing differentially expressed genes in TYLMS-1 treated with Tumour necrosis factor-α-converting enzyme (TACE) inhibitor (TAPI) + γ-secretase inhibitor (DAPT) compared to control TYLMS-1 (dimethyl sulfoxide added). Red and blue dots indicate genes with statistically significant differences (fold-change >1.5, p < 0.05). b Volcano plot showing differentially expressed genes in TYLMS-1 with small interfering (si)EPCAM transfection compared to TYLMS-1 with scramble siRNA transfection. Red and blue dots indicate genes with statistically significant differences (fold-change >1.5, p < 0.05). c – f Conditioned media were prepared by transfection of siEPCAM + siSNAP25, siEPCAM, or scramble siRNA into TYLMS-1 cells. The migration of CD8 + T cells was evaluated and compared using a Transwell assay with conditioned medium. c The expression of EPCAM and SNAP25 in each cell line was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). d Expression of EPCAM and SNAP25 in each cell line was determined using western blotting. e The membrane of the Transwell insert was stained, and the number of migrated CD8 + T cells was evaluated. Representative microscopic images are presented. f Comparison of the number of migrated CD8 + T cells. In each membrane shown in ( e ), the number of stained positive cells was counted in five different fields using a 10× objective lens, and the total number was evaluated. Data are presented as the mean ± standard deviation (SD), n = 3. Two-trailed t -test. * p < 0.05, ** p < 0.01.

Article Snippet: TYLMS-1 and TC616 cells were treated to inhibit epithelial cellular adhesion molecule (EPCAM) signalling with 40 μM tumour necrosis factor-α converting enzyme (TACE) inhibitor (TAPI)-1 (Selleck Chemicals, Houston, TX, USA), 10 μM γ-secretase inhibitor (DAPT) (Selleck Chemicals), or a combination of both inhibitors when the cells reached ~60% confluence.

Techniques: Control, Transfection, Migration, Transwell Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Membrane, Staining, Comparison, Standard Deviation

Journal: Chemotherapy Research and Practice

Article Title: Inhibition of NF- κ B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and γ -Radiation Cytotoxicity in Glioblastoma Cells

doi: 10.1155/2013/593020

Figure Lengend Snippet: (a) The analysis of the methylation status of the MGMT gene in GBM cell lines revealed two groups: methylated (U87MG, U343MG-a, and LN319) and hemimethylated (U251, T98G and U138MG); however, only T98G and U138MG express this gene as detected by real time quantitative PCR (*a pool of 5 white matter samples was used as calibrator); (b) treatment with DHMEQ efficiently decreases the expression of MGMT after 24 h. Data represents two independent experiments in duplicate and are expressed as mean ± SEM; (c) comet assay showed that TMZ-induced DNA damage significantly increases in T98G and U138MG cells as a probable consequence of reduced MGMT expression after exposure to DHMEQ. Each value represents the mean derived from at least three individual experiments (mean ± SD).

Article Snippet: Real-time RT-PCR reactions were performed in triplicate in 10 μ L reactions using the inventoried TaqMan probes (Applied Biosystems, Foster City, CA, EUA) for BCL2 (Hs00608023_m1), BCL-XL (Hs00236329_m1), XIAP (Hs01597783_m1), MMP-2 (Hs01548727_m1), MMP-14 (Hs00237119_m1), uPA (Hs01547054_m1), TIMP-2 (Hs00234278_m1), and MGMT (Hs01037698_m1), on the ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA, EUA).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Expressing, Single Cell Gel Electrophoresis, Derivative Assay

Journal: Epigenetics

Article Title: Radiation-induced epigenetic DNA methylation modification of radiation-response pathways

doi: 10.4161/epi.25498

Figure Lengend Snippet: Table 1. Common differentially methylated genes

Article Snippet: Real time RT-PCR was performed using Taqman Gene Expression Assays (Life Technologies) for KRas (Hs00364284_g1), IGF1R (Hs00609566_m1), and RB1 (Hs01078066_m1) on an Applied Biosystems 7500 Fast Real-time PCR System.

Techniques: Methylation

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Relative expression of mRNA and protein levels of ERCC6 in LECs of controls and ARNCs. a qRT-PCR analysis of the mRNA expression of ERCC6 in LECs of controls and ARNCs. The mRNA levels were normalized by using the GAPDH as the inner control. Data were depicted as the mean ± SD of three independent experiments. * P < 0.01. b The amount of ERCC6 protein in LECs of controls and ARNCs was measured by Western bolt analysis (Samples labeled as #1–#15 of the controls and #1–#15 of the ARNCs out of total 30 samples in each group). c Relative ERCC6 protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Labeling

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Relative expression of mRNA and protein level of DNMTs in LECs of controls and ARNCs. a qRT-PCR analysis of the mRNA expression of DNMTs in LECs of controls and ARNCs. The mRNA levels were normalized by using the GAPDH as the inner control. Values represent mean ± SD. * P < 0.01. b Western bolt analysis for protein level of DNMT3b in LECs of controls and ARNCs (Samples labeled as #1–#15 of the controls and #1–#15 of the ARNCs out of total 30 samples in each group). c Relative DNMT3b protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Labeling

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Protein level and promoter methylation status (site 8) of ERCC6 in HLE-B3 after treatment with UVB exposure. a 12 h after treatment with UVB exposure, the promoter of ERCC6 in the cells displayed hypermethylation compared to the control cells. * P < 0.01. b Protein levels of ERCC6 in control cells and in cells after treatment with UVB exposure. c Relative ERCC6 protein level to GAPDH is presented as mean ± SD. * P < 0.01. d Protein level of active caspases-3 in control cells and HLE-B3 cells after expose to UVB was measured by Western bolt analysis. e Relative active caspases-3 protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Methylation, Control, Western Blot

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Relative mRNA expression and protein levels of ERCC6 in HLE-B3 after treatment with 5-aza-dC. a mRNA expression of ERCC6 in control cells and in cells after treatment with 5-aza-dC for 48 h. b Protein level of ERCC6 in control cells and in cells after treatment with 5-aza-dC. c Relative ERCC6 protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Control

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Knockdown of endogenous Sp1 decreased the mRNA expression and protein level of ERCC6 and Sp1 in 239T cells. a Detection of ERCC6 and Sp1 mRNA expression by qRT-PCR. Values represent mean ± SD. * P < 0.01. b Protein level of ERCC6 and Sp1 was examined by Western blot analysis. Results are presented as mean ± SD. c Relative ERCC6 protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot

Journal: Clinical Epigenetics

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

doi: 10.1186/s13148-016-0229-y

Figure Lengend Snippet: Relative mRNA expression and protein levels of ERCC6 in HLE-B3 after treatment with MS275. a Detection of ERCC6 mRNA expression by qRT-PCR at different time point. b Protein level of ERCC6 was examined by Western blot analysis at different time point. c Relative ERCC6 protein level to GAPDH is presented as mean ± SD. * P < 0.01

Article Snippet: GAPDH (Hs99999905_m1) was used as an internal control. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Biomedicines

Article Title: DNMT3B System Dysregulation Contributes to the Hypomethylated State in Ischaemic Human Hearts

doi: 10.3390/biomedicines10040866

Figure Lengend Snippet: Clinical characteristics of ischaemic cardiomyopathy patients.

Article Snippet: The resulting cDNA was used as a template for RT-qPCR, which was performed in duplicate using the high-performance ViiATM 7 Real-Time PCR System thermal cycler, according to the manufacturer’s instructions (Applied Biosystems; Waltham, MA, USA) and using TaqMan ® (Thermo Fisher Scientific; Horsham, UK) probes: DNMT3B (Hs00171876_m1) and GAPDH (Hs99999905_m1).

Techniques: Sequencing, Biomarker Discovery

Journal: Biomedicines

Article Title: DNMT3B System Dysregulation Contributes to the Hypomethylated State in Ischaemic Human Hearts

doi: 10.3390/biomedicines10040866

Figure Lengend Snippet: mRNA relative expression levels of altered genes involved in the DNA methylation process in ischaemic hearts versus controls. ( a ) Methylation machinery of DNA: genes related to addition ( DNMT3B ), elimination ( TET1 and SMUG1 ) and maintenance ( MBD2 and UHRF1 ) of methyl groups to DNA. ( b ) Methylation machinery regulators ( AHCY , TRAF6 , GADD45G and OGT ). The results were obtained by mRNA-sequencing SOLiD 5500XL platform. Data are presented as the mean ± SEM. a.u., arbitrary units. Ischaemic cardiomyopathy patients ( n = 13; blue), controls subjects ( n = 10; grey). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The resulting cDNA was used as a template for RT-qPCR, which was performed in duplicate using the high-performance ViiATM 7 Real-Time PCR System thermal cycler, according to the manufacturer’s instructions (Applied Biosystems; Waltham, MA, USA) and using TaqMan ® (Thermo Fisher Scientific; Horsham, UK) probes: DNMT3B (Hs00171876_m1) and GAPDH (Hs99999905_m1).

Techniques: Expressing, DNA Methylation Assay, Methylation, Sequencing

Journal: Biomedicines

Article Title: DNMT3B System Dysregulation Contributes to the Hypomethylated State in Ischaemic Human Hearts

doi: 10.3390/biomedicines10040866

Figure Lengend Snippet: DNMT3B system dysregulation. ( a ) DNMT3B mRNA levels in ischaemic hearts ( n = 14) and controls ( n = 8), using RT-qPCR. ( b ) miR-133a-3p expression levels in ischaemic hearts ( n = 22) and controls ( n = 8), using HiSeq 2500 platform. ( c ) miRNAs expression levels in ischaemic hearts ( n = 22) and controls (n = 8), using HiSeq 2500 platform. ( d ) Dnmt3b nuclear protein levels in ischaemic hearts ( n = 8) and controls ( n = 8), using ELISA. ( e ) SAM/SAH ratio in ischaemic hearts ( n = 30) and controls ( n = 6), using ELISA. ( f ) lncRNA H19 expression level in ischaemic hearts ( n = 22) and controls ( n = 8), using the Illumina HiSeq 2500 platform. ( g ) Dnmts nuclear activity in ischaemic hearts ( n = 8) and controls ( n = 8), using ELISA. Data are presented as the mean ± SEM. a.u., arbitrary units. Ischaemic cardiomyopathy patients (blue), controls subjects (grey). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The resulting cDNA was used as a template for RT-qPCR, which was performed in duplicate using the high-performance ViiATM 7 Real-Time PCR System thermal cycler, according to the manufacturer’s instructions (Applied Biosystems; Waltham, MA, USA) and using TaqMan ® (Thermo Fisher Scientific; Horsham, UK) probes: DNMT3B (Hs00171876_m1) and GAPDH (Hs99999905_m1).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 1. Expression of RSK4 in breast cancer tissue. Levels of RSK4 mRNA, as measured by reverse‑transcription quantitative PCR, were compared (A) between breast cancer and paired adjacent normal tissues, as well as (B) between ER+ and ER‑ breast cancer tissues. (C) and RSK4 protein expression in breast cancer and paired adjacent normal tissues, as assessed by immunohistochemistry. Magnification, x400. (D) Bisulfite map showing RSK4 promoter methylation in breast cancer and paired adjacent normal tissues. (E) RSK4 promoter methylation levels in ER+ and ER‑ tumor tissue. Black circles indicated methylated CpGs and white circles presented unmethylated CpGs. Five clones were sequenced per locus. *P<0.05 vs. Normal or ER+. RSK4, X‑linked ribo- somal S6 kinase 4; ER, estrogen receptor.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Methylation, Clone Assay

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 2. Expression of RSK4 in breast cancer cell lines. (A) Western blot analysis of RSK4 protein levels in MCF‑7, MDA‑MB‑231 and MDA‑MB‑453 cells. (B) Bisulfite map showing RSK4 promoter methylation in MCF‑7, MDA‑MB‑231 and MDA‑MB‑453 cells. Black circles indicated methylated CpGs and white circles presented unmethylated CpGs. Five clones per locus were sequenced. *P<0.05. RSK4, X‑linked ribosomal S6 kinase 4; X, no available data.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Methylation, Clone Assay

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 3. RSK4 OE in MCF‑7 cells 72 h following transfection. (A) Bright field (upper row) and fluorescence (lower row) micrographs of MCF‑7 cells trans- fected with RSK4‑OE or NC. Magnification, x400. (B) RSK4 mRNA expression levels in MCF‑7 cells transfected with RSK4‑OE or NC, and in nontransfected controls (blank). Expression values were normalized to the blank group. (C) RSK4 protein levels in MCF‑7 cells transfected with lenti‑RSK4‑EGFP or NC, and in nontransfected controls (blank). β‑actin was used as a loading control. *P<0.05 vs. NC and blank. RSK4, X‑linked ribosomal S6 kinase 4; NC, negative control; OE, overexpression.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Transfection, Fluorescence, Expressing, Control, Negative Control, Over Expression

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 4. Effects of RSK4 OE on the proliferation of MCF‑7 breast cancer cells. (A) Growth curves of MCF‑7 cells transfected with RSK4‑OE or NC, and nontransfected controls (blank), as measured by a Cell Counting Kit‑8 assay. (B) Plates of clone formation in MCF‑7 cells transfected with RSK4‑OE or NC, and blank cells. (C) Quantitation of the experiments. Results are presented as the mean ± standard deviation. *P<0.05 and ***P<0.001 vs. NC and blank. RSK4, X‑linked ribosomal S6 kinase 4; NC, negative control; OE, overexpression.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Transfection, CCK-8 Assay, Quantitation Assay, Standard Deviation, Negative Control, Over Expression

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 5. Effects of RSK4 OE on the migration of MCF‑7 breast cancer cells. (A) images of migratory MCF‑7 cells transfected with RSK4‑OE or NC, and nontransfected controls (blank). Magnification, x100. (B) Quantitation of the experiments. Results are presented as the mean ± standard deviation. *P<0.05 vs. NC and blank. RSK4, X‑linked ribosomal S6 kinase 4; NC, negative control; OE, overexpression.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Migration, Transfection, Quantitation Assay, Standard Deviation, Negative Control, Over Expression

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 6. Effects of RSK4 OE on apoptosis level in MCF‑7 breast cancer cells. (A) Annexin V‑APC fluorescence in MCF‑7 cells transfected with RSK4‑OE or NC, and nontransfected controls (blank). (B) Quantification of the experiments. Results are presented as the mean ± SD. *P<0.05 vs. NC and blank. APC, allophycocyanin; RSK4, X‑linked ribosomal S6 kinase 4; NC, negative control; OE, overexpression; PI, propidium iodide.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Fluorescence, Transfection, Negative Control, Over Expression

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 7. Effects of conditioned medium from MCF‑7 overexpressing RSK4 on HUVEC tube formation. HUVECs were exposed to conditioned medium from MCF‑7 cells transfected with RSK4‑OE or NC, and nontransfected controls (blank). (A) Representative photomicrographs. Magnification, x400. (B) Quantification of tubule number from the experiments (A) Results are presented as the mean ± standard deviation. *P<0.05 vs. NC and blank. RSK4, X‑linked ribosomal S6 kinase 4; HUVECs, human umbilical vein endothelial cells; NC, negative control; OE, overexpression.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Transfection, Standard Deviation, Negative Control, Over Expression

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 8. Effects of estrogen stimulation on ER and RSK4 expression and RSK4 promoter methylation in MCF‑7 cells. (A‑C) ER and RSK4 protein levels in MCF‑7 cells treated with the indicated estrogen concentrations for 48 h. (D) Correlation analysis between ER and RSK4. (E) Bisulfite map showing RSK4 promoter methylation in MCF‑7 cells treated with the indicated estrogen concentrations for 48 h. Black circles indicate methylated CpGs and white circles unmethylated CpGs. Five clones per locus were sequenced. *P<0.05. ER, estrogen receptor; RSK4, X‑linked ribosomal S6 kinase 4.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Expressing, Methylation, Clone Assay

Journal: Oncology reports

Article Title: RSK4 inhibits breast cancer cell proliferation and invasion in vitro, and is correlated with estrogen receptor upregulation in breast cancer.

doi: 10.3892/or.2019.7328

Figure Lengend Snippet: Figure 9. Association between RSK4 promoter methylation level and disease‑free survival in 60 patients with breast cancer. Patients were stratified based on the mean methylation level. RSK4, X‑linked ribosomal S6 kinase 4.

Article Snippet: Following three washes with TBST, the membranes were incubated with rabbit HRP secondary antibodies against RSK4 and ER (1:3,000; Jackson ImmunoResearch Laboratories Inc.), and GAPDH, β-actin and tubulin) (1:3,000; Jackson ImmunoResearch Laboratories Inc.) for 1 h at room temperature.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin-1 inhibits glucocorticoid receptor turnover and alters glucocorticoid signaling

doi: 10.1074/jbc.RA118.007150

Figure Lengend Snippet: β-arrestin-1 influences the GR transcriptional response. Quantitative real-time PCR to measure the expression levels of GR target genes TSC22D3 (A), IL-1β (B), and CXCL5 (C), in cells transfected with NTC or ARRB1-KD siRNA or that were treated with 100 nm dexamethasone for the indicated times. All data represent mean ± S.D. (error bars) from three independent experiments. ****, p < 0.0001 for Dex versus control. #, p < 0.05; ##, p < 0.01; ####, p < 0.0001 to compare ARRB1-KD with NTC at each time point.

Article Snippet: Dexamethasone (chemical name: 1,4-pregnadien-9α-fluoro-16α-methyl-11β,17,21-triol-3,20-dione) and mifeptistone (RU-486) were purchased from Steraloids (Newport, RI).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin-1 inhibits glucocorticoid receptor turnover and alters glucocorticoid signaling

doi: 10.1074/jbc.RA118.007150

Figure Lengend Snippet: PELI1 is differentially regulated by Dex in β-arrestin-1 KD cells. A, quantitative RT-PCR analysis of ARRB1 mRNA (left) and β-arrestin-1 protein (right) levels from A549 cells transfected with NTC or ARRB1-KD siRNA that were treated with vehicle or 100 nm Dex for 3 h. B, qRT-PCR analysis of GR mRNA (left) and protein (right) levels from NTC and ARRB1-KD cells that were treated with vehicle or 100 nm Dex for 3 h. C, NTC and ARRB1-KD cells were treated with vehicle (Ctrl) or 100 nm Dex for 3 h to evaluate GR cellular localization by immunofluorescence. D, quantitative real-time PCR to measure the expression levels of E3 ubiquitin ligase genes in cells transfected with NTC or ARRB1-KD siRNA that were treated with 100 nm dexamethasone for 3 h. Data represent the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 for Dex versus control. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 to compare ARRB1-KD with NTC.

Article Snippet: Dexamethasone (chemical name: 1,4-pregnadien-9α-fluoro-16α-methyl-11β,17,21-triol-3,20-dione) and mifeptistone (RU-486) were purchased from Steraloids (Newport, RI).

Techniques: Quantitative RT-PCR, Transfection, Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, Ubiquitin Proteomics, Control

Journal: Aging (Albany NY)

Article Title: Exosomes as nanostructures deliver miR-204 in alleviation of mitochondrial dysfunction in diabetic nephropathy through suppressing methyltransferase-like 7A-mediated CIDEC N6-methyladenosine methylation

doi: 10.18632/aging.205535

Figure Lengend Snippet: ADSC-derived exosomal miR-204 alleviates HG-induced injury in HK-2 cells. ( A ) Determination of ADSC-derived Exos taken up by HK-2 cells (human proximal renal tubular epithelial cell line) using PKH67 staining. ADSCs were pre-treated with GW4869 (an Exo inhibitor; 2.5 μM), followed by Exo isolation. HK-2 cells were then co-cultured with PKH67-labeled Exos to evaluate their capacity to take up Exos. ( B ) The expression of miR-204 in HK-2 cells using qRT-PCR. ( C , D ) HK-2 cell viability (at 24 h) and apoptosis assessed using CCK-8 and flow cytometry, respectively. ( E ) The levels of MDA, SOD, GPX, and CAT in HK-2 cells using ELISA. ( F ) The expression of Drp1, Fis1, OPA1, and Mfn1 in HK2 cells using western blotting. ( B – F ) HK-2 cells were exposed to HG (30 mM) and cultured for 24 h to construct an in vitro model of DN. To determine the function of exosomal miR-204 in DN, ADSCs were transfected with miR-204 mimic or miR NC (negative control) for 48 h. Then, Exos were separated from the transfected ADSCs and co-cultured with HK-2 cells for 12 h at the concentration of 100 μg/mL. Data were expressed as mean ± standard deviation. ** p < 0.01 vs. Control group; # p < 0.05 and ## p < 0.01 vs. HG group; & p < 0.05 and && p < 0.01 vs. HG + Exo-miR NC group. Abbreviations: ADSC: adipose-derived stem cell; miR-204: microRNA-204; HG: high glucose; Exos: exosomes; qRT-PCR: quantitative real-time polymerase chain reaction; CCK-8: cell counting kit-8; MDA: malondialdehyde; SOD: superoxide dismutase; GPX: glutathione peroxidase; CAT: catalase; ELISA: enzyme-linked immunosorbent assay.

Article Snippet: Proximal renal tubular epithelial cell lines, HK2 (human) and NRK-52E (rat), as well as human embryonic kidney cell line, HEK293T, were supplied by American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay, Staining, Isolation, Cell Culture, Labeling, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Construct, In Vitro, Transfection, Negative Control, Concentration Assay, Standard Deviation, Control, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Aging (Albany NY)

Article Title: Exosomes as nanostructures deliver miR-204 in alleviation of mitochondrial dysfunction in diabetic nephropathy through suppressing methyltransferase-like 7A-mediated CIDEC N6-methyladenosine methylation

doi: 10.18632/aging.205535

Figure Lengend Snippet: ADSC-derived Exos modified by miR-204 inhibits METTL7A-medicated m6A methylation in HG-induced HK-2 cells. ( A ) m6A methylation level in HK-2 cells. ( B ) The expression of METTL3, METTL14, and METTL7A in HK-2 cells using western blotting. ( A , B ) HK-2 cells were exposed to HG (30 mM) and cultured for 24 h to induce an in vitro model of DN. To figure out the impact of exosomal miR-204 in m6A methylation in DN, ADSCs were transfected with miR-204 mimic or miR NC (negative control) for 48 h. Next, Exos were isolated from the transfected ADSCs and co-cultured with HK-2 a for 12 h at the concentration of 100 μg/mL. Data were expressed as mean ± standard deviation. ** p < 0.01 vs. Control group; ## p < 0.01 vs. HG group; && p < 0.01 vs. HG + Exo-miR NC group; ns indicates no significant differences between groups. ( C , D ) The expression of miR-204 and METTL7A in HEK293T cells using qRT-PCR and western blotting. ( C , D ) Data were expressed as mean ± standard deviation. ** p < 0.01 vs. mimic NC group; ^^ p < 0.01 vs. inhibitor NC group. ( E ) Determination of the interaction between miR-204 and METTL7A in HEK293T cells using dual-luciferase reporter assay. Data were expressed as mean ± standard deviation. ** p < 0.01 vs. miR NC group; ns indicates no significant differences between groups. Abbreviations: ADSC: adipose-derived stem cell; Exos: exosomes; miR-204: microRNA-204; METTL: methyltransferase-like; m6A: N6-methyladenosine; HG: high glucose; qRT-PCR: quantitative real-time polymerase chain reaction.

Article Snippet: Proximal renal tubular epithelial cell lines, HK2 (human) and NRK-52E (rat), as well as human embryonic kidney cell line, HEK293T, were supplied by American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay, Modification, Methylation, Expressing, Western Blot, Cell Culture, In Vitro, Transfection, Negative Control, Isolation, Concentration Assay, Standard Deviation, Control, Quantitative RT-PCR, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction

Journal: Aging (Albany NY)

Article Title: Exosomes as nanostructures deliver miR-204 in alleviation of mitochondrial dysfunction in diabetic nephropathy through suppressing methyltransferase-like 7A-mediated CIDEC N6-methyladenosine methylation

doi: 10.18632/aging.205535

Figure Lengend Snippet: ADSC-derived Exos modified by miR-204 prevent DN progression via downregulating METTL7A expression in HG-induced HK-2 cells. ( A , B ) The expression of miR-204 and METTL7A in HK-2 cells using qRT-PCR and western blotting. ( C , D ) HK-2 cell viability (at 24 h) and apoptosis using CCK-8 and flow cytometry, respectively. ( E ) The levels of MDA, SOD, GPX, and CAT in HK-2 cells using ELISA. Exos were isolated from either miR-204 mimic- or miR NC-transfected ADSCs, which were subsequently co-cultured with HG-induced HK-2 cells for 12 h at the concentration of 100 μg/mL. Furthermore, HG-induced HK-2 cells were partially transected with oe-METTL7A and co-cultured with Exos from miR-204 mimic-transfected ADSCs to ascertain the link between miR-204 and METTL7A in DN. Data were expressed as mean ± standard deviation. ## p < 0.01 vs. HG + Exo-miR NC group; ^^ p < 0.01 vs. HG + Exo-miR-204 mimic group; ns indicates no significant differences between groups. Abbreviations: ADSC: adipose-derived stem cell; Exos: exosomes; miR-204: microRNA-204; DN: diabetic nephropathy; METTL: methyltransferase-like; HG: high glucose; qRT-PCR: quantitative real-time polymerase chain reaction; CCK-8: cell counting kit-8; MDA: malondialdehyde; SOD: superoxide dismutase; GPX: glutathione peroxidase; CAT: catalase; ELISA: enzyme-linked immunosorbent assay.

Article Snippet: Proximal renal tubular epithelial cell lines, HK2 (human) and NRK-52E (rat), as well as human embryonic kidney cell line, HEK293T, were supplied by American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay, Modification, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Isolation, Transfection, Cell Culture, Concentration Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Aging (Albany NY)

Article Title: Exosomes as nanostructures deliver miR-204 in alleviation of mitochondrial dysfunction in diabetic nephropathy through suppressing methyltransferase-like 7A-mediated CIDEC N6-methyladenosine methylation

doi: 10.18632/aging.205535

Figure Lengend Snippet: METTL7A silencing prevents DN progression by downregulating CIDEC m6A methylation in HG-induced HK-2 cells. ( A , B ) The expression of CIDEC in HK-2 cells using qRT-PCR and western blotting. ( C ) CIDEC m6A methylation level in HK-2 cells using MeRIP-qPCR. ( A – C ) Data were expressed as mean ± standard deviation. # p < 0.05 and ## p < 0.01 vs. HG + si-NC group. ( A – C ) HG-induced HK-2 cells were partially transfected with si-METTL7A or si-NC (negative control) for 48 h to explore the effects of METTL7A on CIDEC and its m6A methylation. ( D ) Determination of the interaction between METTL7A and CIDEC in HEK293T cells using RNA pulldown assay. ( E , F ) The expression of CIDEC in HK-2 cells using western blotting and qRT-PCR. ( G , H ) HK-2 cell viability and apoptosis using CCK-8 and flow cytometry, respectively. ( I ) The levels of MDA, SOD, GPX, and CAT in HK-2 cells using ELISA. ( E – I ) Empty vector (control), si-CIDEC, or/and oe-METTL7A were transfected into HG-induced HK-2 cells to further determine the interaction between METTL7A and CIDEC in DN. Data were expressed as mean ± standard deviation. ## p < 0.01 vs. HG + vector group; ^^ p < 0.01 vs. HG + si-CIDEC group. Abbreviations: METTL: methyltransferase-like; DN: diabetic nephropathy; CIDEC: cell death-inducing DFF45-like effector C; m6A: N6-methyladenosine; HG: high glucose; qRT-PCR: quantitative real-time polymerase chain reaction; MeRIP-qPCR: methylated RNA immunoprecipitation-PCR; CCK-8: cell counting kit-8; MDA: malondialdehyde; SOD: superoxide dismutase; GPX: glutathione peroxidase; CAT: catalase; ELISA: enzyme-linked immunosorbent assay.

Article Snippet: Proximal renal tubular epithelial cell lines, HK2 (human) and NRK-52E (rat), as well as human embryonic kidney cell line, HEK293T, were supplied by American Type Culture Collection (Manassas, VA, USA).

Techniques: Methylation, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Transfection, Negative Control, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Cell Counting

Journal: Aging (Albany NY)

Article Title: Exosomes as nanostructures deliver miR-204 in alleviation of mitochondrial dysfunction in diabetic nephropathy through suppressing methyltransferase-like 7A-mediated CIDEC N6-methyladenosine methylation

doi: 10.18632/aging.205535

Figure Lengend Snippet: ADSC-derived Exos modified by miR-204 rescue OS-induced mitochondrial dysfunction in DN rats by inhibiting METTL7A-mediated CIDEC m6A methylation. ( A , B ) The levels of MDA, SOD, GPX, and CAT in the kidney tissues and plasma of rats in each group using ELISA. ( C , D ) The expression of Drp1, Fis1, OPA1, Mfn1, METTL7A, and CIDEC in the kidney tissues of rats in each group using western blotting. ( E ) CIDEC m6A methylation levels in HK-2 cells using MeRIP-qPCR. Rats in the Exo group were treated with 1.6 mg/kg ADSC-derived Exos via caudal vein injection every two days for eight successive weeks. Meanwhile, rats in the Exo-mimic NC and Exo-miR-204 mimic groups were treated with ADSC-derived Exos loaded with mimic-NC or miR-204 mimic, respectively. Rats in the DN group were injected with the same volume of saline. Data were expressed as mean ± standard deviation ( n = 6/group). * p < 0.05 and ** p < 0.01 vs. Control group; ^ p < 0.05 and ^^ p < 0.01 vs. DN + Exo-mimic NC group. Abbreviations: ADSC: adipose-derived stem cell; Exos: exosomes; miR-204: microRNA-204; OS: oxidative stress; DN: diabetic nephropathy; METTL: methyltransferase-like; CIDEC: cell death-inducing DFF45-like effector C; m6A: N6-methyladenosine; MDA: malondialdehyde; SOD: superoxide dismutase; GPX: glutathione peroxidase; CAT: catalase; ELISA: enzyme-linked immunosorbent assay; MeRIP-qPCR: methylated RNA immunoprecipitation-PCR.

Article Snippet: Proximal renal tubular epithelial cell lines, HK2 (human) and NRK-52E (rat), as well as human embryonic kidney cell line, HEK293T, were supplied by American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay, Modification, Methylation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Injection, Saline, Standard Deviation, Control, RNA Immunoprecipitation