Journal: The Journal of Biological Chemistry

Article Title: Nascent RNA Cleavage by Arrested RNA Polymerase II Does Not Require Upstream Translocation of the Elongation Complex on DNA *

doi:

Figure Lengend Snippet: A, time course of SII-dependent elongation from site Ia in the presence of all four nucleotides. Washed complexes (lane Ia) were split into 2 aliquots. One received bovine brain SII and 7 mM MgCl2 and was incubated at 28 °C for 1.5 or 15 min to generate the first (*) and second (**) cleavage intermediates, respectively. The second aliquot of washed complexes received bovine brain SII, MgCl2, and 800 μM each of all four NTPs. Portions of this reaction were stopped after the indicated times at 28 °C and analyzed by electrophoresis with the first and second cleavage intermediates. RO, runoff RNA. B, RNA elongation by an SII-independent elongation complex in the presence of SII. RNA in washed complexes was extended for 10 min at 28 °C to positions G218/G220 (indicated by dash at left, lane 0) in the presence of UTP, CTP, and GTP (800 μM each), bovine brain SII, and 7 mM MgCl2. The reaction was chilled to 4 °C, ATP (800 μM) was added, and samples were stopped at the indicated times after incubation at 28 °C. One sample (sar) was adjusted to 0.25% in Sarkosyl and another (α) to 1 μg/ml in α-amanitin before the addition of ATP and incubation at 28 °C. Arrowheads indicate the position of marker RNAs of 260, 380, 420, and 540 nucleotides (bottom to top). C, RNA elongation by a second SII-independent elongation complex in the presence of SII. Elongation complexes were assembled at site Ia (Ia) and moved to positions G218/G220 (dash to left of figure) as described in the legend to B. These complexes were washed free of nucleotides by centrifugation and resuspension and moved to position C230 (U) after an 8-min incubation at 28 °C in the presence of bovine brain SII, 7 mM MgCl2, and 800 μM each of ATP, GTP, and CTP. The reaction was incubated at 28 °C with UTP (800 μM) for the indicated times. One sample (sar) was made 0.25% in Sarkosyl before the addition of UTP and incubation at 28 °C.

Article Snippet: Fast protein liquid chromatography-purified NTPs, 4 ddNTPs, and 3′- O -methyl GTP were purchased from Pharmacia LKB Biotechnology Inc., 3′-dUTP was purchased from Boehringer Mannheim.

Techniques: Incubation, Electrophoresis, Marker, Centrifugation

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: Cell Reports

Article Title: Axonal Odorant Receptors Mediate Axon Targeting

doi: 10.1016/j.celrep.2019.11.099

Figure Lengend Snippet:

Article Snippet: Protein concentration was determined using BCA protein concentration assay kit (Thermo Scientific), following the manufacturers’ instructions.

Techniques: Marker, Recombinant, Protein Concentration, Reverse Transcription, Software, Fast Protein Liquid Chromatography, Mass Spectrometry

Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: PNGase F-released N-glycans were subject to solid phase extraction, whereby the neutral-enriched fraction was eluted with 40% acetonitrile, prior to fluorescent labelling and chromatography on an RP-amide column; each fraction was collected and subject to MALDI-TOF MS (m/z values for [M+H]+ being indicated); the glycans in each fraction are shown in order of occurrence with the most dominant glycan uppermost. The annotations in the Symbolic Nomenclature for Glycans (see also key in grey box) are based on elution time, MS/MS and digestion data in comparison to recently-published data on royal jelly, mosquito and moth N-glycans; for some simple glycans, a table of elution times in comparison to previous studies is given in Supplementary Table 2, whereby the order of retention is generally consistent with that tabulated by Tomiya (49). The glycome is dominated by typical insect glycans (oligomannosidic, paucimannosidic and hybrid), but also some complex bi-/tri-antennary forms are present; two phosphoethanolamine (PE)-modified glycans in this pool are highlighted in the blue boxes and fucosylated oligomannosidic structures are in light grey boxes. The column was calibrated in terms of glucose units (g.u.). For the core α1,3-fucosylated N-glycans released with PNGase A from larval glycopeptides, refer to Supplementary Figure 1.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification

Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: N-glycans released by the combined use of PNGase F and Ar were subject to solid phase extraction, whereby the neutral-enriched fraction was eluted with 40% acetonitrile, prior to fluorescent labelling and chromatography on an RP-amide column; each fraction was collected and subject to MALDI-TOF MS. The annotations in the Symbolic Nomenclature for Glycans (see also key in grey box) are based on elution time, MS/MS and digestion data (see examples in Supplementary Figures 2 and 3). The column was calibrated in terms of glucose units. Phosphoethanolamine (PE)- or α-GalNAc-modified glycans in this pool are highlighted respectively in blue or green boxes, structures previously found on honeybee venom phospholipase A2 and hyaluronidase are in light grey boxes and those hybrid or biantennary forms reported by us in royal jelly in light yellow boxes; seven different isomers of Hex3HexNAc4Fuc2 are indicated by the m/z 1687 values in red. Due to their low abundance, neither the biantennary and Man4-5GlcNAc2-based hybrid glycans nor the PE-, β1,3-Gal and α1·4· alNAc-modified antennae were previously detected in honeybee venom.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification

Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: PNGase F/Ar-released N-glycans were subject to solid phase extraction, whereby the anionic-enriched fraction was eluted with 40% acetonitrile/0.1% trifluoroacetic acid, prior to fluorescent labelling and chromatography on an RP-amide column; each fraction was collected and subject to MALDI-TOF MS. The annotations are based on elution time, MS/MS and digestion data (see Supplementary Figure 6); greyscale structures indicate the elution times of co-fractionating neutral glycans. The column was calibrated in terms of glucose units. Glycans with HexNAc3- or glucuronylated/phosphoethanolamine-modified antennae are highlighted respectively in green and blue boxes.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification

Journal: The EMBO Journal

Article Title: Limited oxygen in standard cell culture alters metabolism and function of differentiated cells

doi: 10.1038/s44318-024-00084-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Maintenance of iPSCs: Human induced pluripotent stem cells (iPSCs) were maintained on vitronectin XFTM (10 μg/mL, StemCell Technologies)-coated plates and in Essential 8 (E8) medium consisting of DMEM/F12 (Gibco), l -ascorbic acid 2-phosphate (1%), insulin-transferrin-selenium solution (2%, Life Technologies), sodium bicarbonate (0.7%), and Penicillin/Streptomycin (P/S) (1%), freshly supplemented with TGFβ (10 ng/mL, R&D) and FGF2 (12 ng/mL, Qkine).

Techniques: Recombinant, Affinity Purification, Sequencing, Saline, Bicinchoninic Acid Protein Assay, Reverse Transcription, SYBR Green Assay, Software, Imaging, Gas Chromatography, Mass Spectrometry, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on total levels of proteins that regulate mitochondrial dynamics. (A) Mfn1 and (B) DRP1 western blot of lysates from PC12 cells treated with FCCP (10 μM, 2 h) and AβOs (0.5 μM, 1 and 2 h). A dotted line on the western blot indicates different regions of the same gel. (C, D) Quantification of Mfn1 and DRP1 levels normalized to beta actin. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 compared between the control group. One-way ANOVA with the Dunnett's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n = 4–5 for each group) (original gel blot are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on immunoreactivity of proteins that regulate mitochondrial dynamics. Representative epifluorescence images of (A) Mfn1 and (B) DRP1 immunoreactivity in PC-12 cells control and treated with AβOs (0.5 μM) for 1 and 2 h. Quantification of (C) Mfn1 and (D) DRP1 immunoreactivity (intensity), under the same experimental conditions. Scale bars: 20 μm. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001 compared between the control group. One-way ANOVA with the Dunn's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n=3–6 for each group, N= 56–103) (entire inmunocytochemistry for Mfn1 and DRP1 with control are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Model of alterations induced on SIRT1/PGC-1α pathway by AβOs. (A) . Healthy neuronal conditions were energy depletion and/or decreased catabolic rates can be sensed by SIRT1 promoting the PGC-1α deacetylation (1). Transcription and co-activates of factors like NRF- 1/2 for the expression of nuclear-encoded mitochondrial genes and dynamic mitochondrial proteins, requires of PGC-1α translocation (2). Coordinated mitochondrial dynamics (3, fission/fusion), depends of adequate expression of Mfn1, Drp1. (B) . In AβOs treated neurons, the PGC-1α is unable to be deacetylated and to translocate to the nucleus (4). The expression of key genes is loss (5), and imbalance between fusion and fission to promote the granular mitochondrial phenotype (6) and neuronal death.

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Expressing, Translocation Assay

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 1. High glucose increases O-GlcNAcylation of DNMT1 in cell lines and primary cells. (A) Hep3B cells were treated with glucose (5 mM or 25 mM) with or without Thiamet-G (TMG). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). (B) Lysates of Hep3B treated with glucose were immunoprecipitated with DNMT1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc (n = 3). (C) Peripheral blood mononuclear cells (PBMCs) were isolated from three individual donor blood samples and treated with increasing concentration of glucose for 24 hr. Collected cell lysates from PBMCs were immunoprecipitated with antibody targeting DNMT1 and immunoblotted for O-GlcNAc. Representative blot from one donor (n = 3). (D) Immunoblots for O-GlcNAc and GAPDH from liver samples of C57BL/6J mice given a high- fat/high-sucrose diet (HF/HS) or normal diet (chow) for 4 mo, and immunoprecipitated with Dnmt1. Lysates of mouse liver were immunoprecipitated with Dnmt1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc. *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A-D); ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Western Blot, Immunoprecipitation, Isolation, Concentration Assay

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 2. Identification of O-GlcNAcylated sites within DNMT1 by LC-MS/MS. (A) Schematic drawing of the DNMT1 O-GlcNAc-modified region enriched from Hep3B cells based on mass spectrometry (MS) data and tandem MS (MS/MS) peaks. FTMS+ p NSI full MS (400.0000–1600.0000). DQDYARFESPPKTQPTEDNKF (S9 HexNAc) – S878. (B) Schematic diagram of identified novel O-GlcNAcylated and phosphorylated sites within DNMT1 as determined via LC-MS/MS. DMAP, DNA methyltransferase associated protein-binding domain; PCNA, proliferating cell nuclear antigen-binding domain; NLS, nuclear localization sequences; RFTS, replication foci targeting sequence domain; BAH, bromo-adjacent homology domain. (C) Sequence conservation of S878 in vertebrates. (D) Each immunoprecipitated Myc-DNMT1 wild type and substituted mutants was immunoblotted with an O- GlcNAc antibody (n = 3). **p<0.0005; ***p<0.0001 by Student’s t-test (D); N.D., not detected, ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Liquid Chromatography with Mass Spectroscopy, Modification, Mass Spectrometry, Tandem Mass Spectroscopy, Protein Binding, Binding Assay, Sequencing, Immunoprecipitation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 4. High glucose leads to loss of DNA methylation at cancer-specific partially methylated domains (PMDs). (A) Density plot of DNA methylation for DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Genome browser screenshot of DNA methylation for DNMT1-WT and DNMT1-S878A cells and low or high glucose along with liver tumor PMDs from Li et al., 2016. (C) Boxplots of DNA methylation at PMDs or general genomic background (BG) for each DNMT1-WT and DNMT1-S878A treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). (D) Heatmap representation of global DNA methylation for DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc) at gene-poor and gene-rich regions. (E) Methylation changes from O-GlcNAcylation of DNMT1 by wave score for replication timing (Hansen et al., 2010; Thurman et al., 2007). ***p<0.0001 by Wilcoxon signed-rank test (C).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: DNA Methylation Assay, Methylation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 5. High glucose-induced reactive oxygen species (ROS) and DNA damage cause apoptotic cell death in DNMT1-WT cells. (A) Quantitative fluorescence image of ROS in DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Quantitative fluorescence image of γ-H2A.X in DNMT1-WT and DNMT1-S878A cells treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O- GlcNAc). (C) Quantitative fluorescence image of cell death in propidium iodide staining of DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A–C); data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Fluorescence, Staining