Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: bioRxiv

Article Title: Human endometrial KISS1R inhibits stromal cell decidualization in a manner associated with a reduction in ESR1 levels

doi: 10.1101/2022.11.20.517219

Figure Lengend Snippet: Quantification of mRNA levels of hallmark molecular markers of decidualization (A) FOXO1 , (B) IGFBP1 , (C) PRL and ( D ) representative western blot analysis of endometrial FOXO1 protein levels with tubulin serving as the housekeeping control before and after six days of decidualization (see Fig. S2 for additional blots). Quantification of mRNA levels of known regulators of ESC decidualization ( E ) DCN , ( F ) LEFTY2 , and ( G ) WNT4 before and after six days of decidualization. qPCR analysis of mRNA expression of ( H ) KISS1 and ( I and J ) KISS1R before and after one, two, four, and six days of decidualization. Statistics (biological replicates: n=3-6 biopsies and technical replicates: n=3-6 for each group): student’s unpaired two-tailed t-test and one-way ANOVA with Bonferroni Multiple Comparison post hoc test. * p<0.05; ** p<0.01; and *** p<0.001. D0: day zero; D1: day one; D2: day two; D4: day four; D6: day six of decidualization; BM: basal medium; and EPC: decidualization medium.

Article Snippet: The amplified cDNA was diluted to 10 ng/μl, and quantitative PCR was performed using 40 ng of cDNA with the following TaqMan probes (Applied Biosystems/Life Technologies), KISS1 (Hs00560041_m1), and KISS1R (Hs00173906_m1).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Human endometrial KISS1R inhibits stromal cell decidualization in a manner associated with a reduction in ESR1 levels

doi: 10.1101/2022.11.20.517219

Figure Lengend Snippet: Quantification of mRNA and protein levels in empty vector control and KISS1R expressing primary HESCs following six days of in vitro decidualization. qPCR analysis of ( A) KISS1 , ( B ) KISS1R and phenotypic markers of decidualization ( C ) DESMIN, KRT7 , and KRT18 mRNA levels. Representative images of the immunofluorescence analysis of cytokeratin 7 protein levels in empty vector control ( D1-3 ) and KISS1R ( D4-6 ) expressing primary HESCs. ( E ) qPCR analysis of decidualization and ESR1 signaling markers FOXO1, IGFBP1, PRL, DCN, LEFTY2, WNT4, ESR1, PGR, MMP2 , and MMP7 mRNA levels. Representative western blot analysis of endometrial FOXO1 and ESR1 protein levels with tubulin serving as the housekeeping control before and after six days of decidualization in empty vector and KISS1R expressing HESCs (see Fig. S3 for additional blots). Statistics (biological replicates: n=3-5 biopsies and technical replicates: n=3-6 for each group): student’s unpaired two-tailed t-test. * p<0.05; ** p<0.01; and *** p<0.001. EV: Empty vector control; KISS1R or KR: KISS1R expression; BM: basal medium; and EPC: decidualization medium.

Article Snippet: The amplified cDNA was diluted to 10 ng/μl, and quantitative PCR was performed using 40 ng of cDNA with the following TaqMan probes (Applied Biosystems/Life Technologies), KISS1 (Hs00560041_m1), and KISS1R (Hs00173906_m1).

Techniques: Plasmid Preparation, Expressing, In Vitro, Immunofluorescence, Western Blot, Two Tailed Test

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: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Exploration of PCIF1 target genes by genome-wide gene expression analysis. ( A ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) or two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2). ( B ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2) with anti-PCIF1 and anti-b-actin antibodies. ( C , D ) Venn diagrams showing the overlap between the two indicated siRNA-mediated downregulated ( C ) and upregulated ( D ) genes identified by the gene expression profile analyzed by DNA microarray using a Human Genome U133 Plus 2.0 Array (Affymetrix). The condition for selecting differentially expressed genes are as follows: (1) cut-off condition: expression > 100, (2) fold change: >2, (3) p -value < 0.05.

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Isolation, Control, Western Blot, Microarray, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Expression of RAB23 and CNOT6 is regulated by PCIF1 at both the mRNA and protein levels. ( A – C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3), using the specific primer set detecting PCIF1 ( A ), RAB23 ( B ), and CNOT6 ( C ) expression. ( D , E ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) with the indicated antibodies. Signal intensities obtained from immunoblotting were quantified using ImageJ software version 1.52. The y-axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Software, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant PCIF1 restores the normal levels of target mRNA expression. ( A , B ) RT-qPCR analysis of total RNAs isolated from HeLa cells transfected with a control empty vector (Vec) or a vector expressing siRNA-resistant PCIF1 (PCIF1siR) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1 #3), using the specific primer set detecting RAB23 ( A ) and CNOT6 ( B ) expression. The y -axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between indicated pairs (Student’s t -test, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 does not regulate expression of its target genes at the transcriptional level. ( A , E ) Schematic illustrations of the RAB23 ( A ) and CNOT6 ( E ) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. ( B – D , F – H ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. ( I , J ) ChIP analyses of the RAB23 ( I ) and CNOT6 ( J ) gene promoters (position 2 in ) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Isolation, Control, Negative Control, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 regulates the stability of target gene mRNAs in opposite ways. After HeLa cells were treated with a negative control siRNA (siNC) and two PCIF1-targeted siRNAs (siPCIF1 #1 and #3) for 72 h, actinomycin D was added to inhibit transcription. Cells were harvested at 0, 2, 4, 8, and 12 h after treatment, and total RNA was isolated. The amount of residual mRNA was analyzed by RT-qPCR at each time point, using the specific primer set detecting PCIF1 ( A ), CRAB23 ( B ), and CNOT6 ( C ) mRNAs. The relative value was calculated using the expression level of β-actin mRNA (ACTB) as a normalizer. The relative values at each time point were calculated relative to time zero. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Negative Control, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant wild-type but not methyltransferase-deficient mutant PCIF1 restored normal levels of target mRNA expression. ( A ) Immunoblotting analysis of total protein extracts from HeLa cells transfected with a control empty vector (Vec), a vector expressing siRNA-resistant wild-type PCIF1 (siR_wt), or methyltransferase-deficient mutant PCIF1 (siR_mut) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression) with the indicated antibodies. ( B , C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated as in ( A ), using the specific primer set detecting RAB23 ( B ) and CNOT6 ( C ) expression. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 suppression resulted in a significant decrease in m 6 A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m 6 A antibody, using the specific primer set detecting RAB23 ( A ), CNOT6 ( B ), and ACTB ( C ) mRNAs. The y -axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m 6 A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Control, Quantitative RT-PCR, Purification, Negative Control, Standard Deviation