Journal: Frontiers in Cellular Neuroscience

Article Title: Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells

doi: 10.3389/fncel.2015.00350

Figure Lengend Snippet: Leptin-triggered cell death is inhibited by shRNA-silencing of cyclin D1 expression in SVZ neurospheres . (A) GFP fluorescence (top row) and cyclin D1 immunocytofluorescence (bottom row) in neurospheres with single GFP transfection in the absence of leptin (first column from left, “control”) or with double GFP- and shRNA cyclinD1- transfection in the absence (second column) or in the presence (third column) of 6.2 nM leptin. (B) Quantification of cyclinD1-immunoreactive cells per neurosphere in the three experimental conditions. (C) Enumeration of cells per neurosphere in the three cultures run in parallel. Data are expressed as mean ± SEM of three independent cultures. * significantly different from control at p < 0.05.

Article Snippet: The sequence of cyclin-specific shRNA of the cyclin D1 (CCND1) gene was designed by using siRNA Target Designer ( www.promega.com/siRNADesigner/program/default.asp ) soft-ware.

Techniques: shRNA, Expressing, Fluorescence, Transfection, Control

Journal: Theranostics

Article Title: The orchestration of cell-cycle reentry and ribosome biogenesis network is critical for cardiac repair

doi: 10.7150/thno.96460

Figure Lengend Snippet: Inhibition of Rb1 expression promotes CMs proliferation. ( A-C ) RT-qPCR, and Western blot analysis of Rb1 expression in CMs transfected with siRNA targeting Rb1 (si-Rb1) or control (si-Control) (n=3). ( D, E ) Flow cytometry analysis of CMs proliferation (n=5). ( F, G ) The proliferation of CMs was detected by EdU incorporation. The cells were pre-treated with si-Control or si-Rb1. Blue: nuclear staining (DAPI); Red: EdU staining (scale bar: 50 μm) (n=4). ( H, I ) The proliferation of CMs was detected by pH3 immunofluorescence. Blue: nuclear staining (DAPI); Red: pH3 staining (scale bar: 50 μm) (n=4). ( J ) CMs from GFP mice were treated with si-Control or si-Rb1 (n=3). Data are presented as the mean±SEM.

Article Snippet: An siRNA designed to target the coding region of Rb1 mRNA (GTCAAGGGCTTACCATACT) was synthesized by Ribobio (Guangzhou, China).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Control, Flow Cytometry, Staining, Immunofluorescence

Journal: Theranostics

Article Title: The orchestration of cell-cycle reentry and ribosome biogenesis network is critical for cardiac repair

doi: 10.7150/thno.96460

Figure Lengend Snippet: circASXL1 promotes Ribo-bio by enhancing Ncl expression. ( A ) The density of circASXL1 binding sites in the designated regions of Ncl mRNA. ( B ) Bioinformatics prediction using the RNAhybrid data base revealed that circASXL1 has a continuous binding site with the 5' UTR of Ncl mRNA. ( C ) The interaction between circASXL1 and Ncl mRNA 5' UTR was confirmed by RNA pulldown assay (n=4). ( D, E ) Western blot analysis of the expression of Ncl protein in CMs transfected with circASXL1 shRNA or overexpression plasmid (n=3). ( F ) RT-qPCR analysis showed that Ncl siRNA treatment led to reduced levels of pre-47S rRNA, 28S, 5.8S transcript (n=3). ( G ) Western blot analysis of the expression of Ncl and Pol Ι protein in CMs transfected with Ncl siRNA (n=3). ( H-J ) rRNA synthesis, proliferation, and cytokinesis of CMs transfected with Ncl siRNA (si-Ncl) or control (si-Control) were determined by EU staining (red), EdU incorporation, and Aurora B staining, respectively (n=5). ( K, L ) The primary CMs from GFP mice were transfected with si-circASXL1 or si-control. Nuclei were identified by DAPI staining. Blue arrow: mononuclear; White arrow: multinucleated (n=3). Data are presented as the mean±SEM.

Article Snippet: An siRNA designed to target the coding region of Rb1 mRNA (GTCAAGGGCTTACCATACT) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Binding Assay, Western Blot, Transfection, shRNA, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Control, Staining

Journal: Molecular Medicine Reports

Article Title: miR-29a-3p represses proliferation and metastasis of gastric cancer cells via attenuating HAS3 levels

doi: 10.3892/mmr.2018.8896

Figure Lengend Snippet: PCR primer sequences.

Article Snippet: To RNA interference, pre-designed validated siRNA targeting miR-29a-3p and HAS3 were obtained from RiboBio (Guangzhou, China) and transfected into cells using a ribo FECTTM CP Transfection kit (RiboBio) following the manufacturer's protocol.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: miR-29a-3p represses proliferation and metastasis of gastric cancer cells via attenuating HAS3 levels

doi: 10.3892/mmr.2018.8896

Figure Lengend Snippet: Bioinformatic target prediction of miR-29a-3p. (A) Bioinformatic target prediction was performed with three online tools and the intersectional potential target genes were shown. A putative miR-29a binding sequence is identified in the HAS3 3′UTR region. (B) Reverse transcription-quantitative polymerase chain reaction analysis of miR-29a-3p expression in GC9811 cells with miR-29a-3p knockdown and GC9811-p cell with miR-29a-3p overexpression. miR, microRNA; si, short interfering RNA; si-NS, non-specific control siRNA.

Article Snippet: To RNA interference, pre-designed validated siRNA targeting miR-29a-3p and HAS3 were obtained from RiboBio (Guangzhou, China) and transfected into cells using a ribo FECTTM CP Transfection kit (RiboBio) following the manufacturer's protocol.

Techniques: Binding Assay, Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Over Expression, Small Interfering RNA, Control

Journal: Molecular Medicine Reports

Article Title: miR-29a-3p represses proliferation and metastasis of gastric cancer cells via attenuating HAS3 levels

doi: 10.3892/mmr.2018.8896

Figure Lengend Snippet: Knockdown of HAS3 suppresses GC9811 cell proliferation and migration. (A) Knockdown of HAS3 by siRNA in GC9811-p was confirmed by western blot analysis. Cell proliferation and mobility were assessed by (B) MTT assay, (C) Transwell migration assay and (D) would healing assay. Magnification, ×100. *P<0.05, **P<0.01 vs. the adjacent control group. si, short interfering RNA; si-NS, non-specific control siRNA.

Article Snippet: To RNA interference, pre-designed validated siRNA targeting miR-29a-3p and HAS3 were obtained from RiboBio (Guangzhou, China) and transfected into cells using a ribo FECTTM CP Transfection kit (RiboBio) following the manufacturer's protocol.

Techniques: Knockdown, Migration, Western Blot, MTT Assay, Transwell Migration Assay, Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: miR-29a-3p represses proliferation and metastasis of gastric cancer cells via attenuating HAS3 levels

doi: 10.3892/mmr.2018.8896

Figure Lengend Snippet: HAS3 knockdown resulted in a decrease in proliferation and metastatic potential of GC9811 cells with silencing of miR-29a-3p. (A) Knockdown of HAS3 by siRNA in GC9811 with silencing of miR-29a-3p was confirmed by western blot analysis. Cell proliferation and mobility were assessed by (B) MTT assay, (C) Transwell migration assay and (D) would healing assay. Magnification, ×100. **P<0.01 vs. the adjacent control group. si, short interfering RNA; si-NS, non-specific control siRNA.

Article Snippet: To RNA interference, pre-designed validated siRNA targeting miR-29a-3p and HAS3 were obtained from RiboBio (Guangzhou, China) and transfected into cells using a ribo FECTTM CP Transfection kit (RiboBio) following the manufacturer's protocol.

Techniques: Knockdown, Western Blot, MTT Assay, Transwell Migration Assay, Control, Small Interfering RNA

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Dynamic profile of lncRNAs in myoblasts and differentiated myotube. (A) RNA dot blot assay of m 6 A methylation levels of C2C12 myoblasts in GM and D4. (B) MA plot shows the relationship of expression abundance and fold changes of lncRNAs in myoblasts (GM) and myotube (D4). log2 (MeanRPKM) represents gene expression values, log2Fold Change represents the fold change of lncRNAs at D4 compared to GM. Red dots represent 582 significantly up-regulated lncRNAs at D4 in relation to GM, adjusted P ≤ 0.05; Green dots, represent 97 significantly down-regulated lncRNAs at D4 in relation to GM, adjusted P ≤ 0.05; Yellow dots represent lncRNA without significantly differential expression, adjusted P > 0.05. Top differentially expressed lncRNAs were marked in purple. (C) The volcano plot shows significantly differentially expressed lncRNAs at GM and D4. Red dots represent 582 significantly up-regulated lncRNAs at D4, adjusted P ≤ 0.05; Green dots, represent 97 significantly down-regulated lncRNAs at D4, adjusted P ≤ 0.05; Yellow dots represent lncRNA without significantly differential expression, adjusted P > 0.05. Top differentially expressed lncRNAs were marked in purple. (D) The Venn diagram shows 97 and 582 significantly differentially expressed lncRNAs in GM and D4, and 4904 lncRNAs without significant difference between GM and D4. (E) The heatmap shows significantly differentially expressed lncRNAs in GM and D4. Yellow color represents up-regulation in D4, while green color represents down-regulation. Row represents genes, column represents samples, and each cell represents expression value. (F) Pie chart show the gene types of lncRNAs up-regulated in GM. (G) Pie chart show the gene types of lncRNAs up-regulated in D4. (H) Quantitative real-time reverse transcription PCR (qRT-PCR) validated top differently expressed lncRNAs in developing muscle cells. RPKM: Reads Per Kilobase per Million mapped reads. adjusted P: adjusted p -value; Data are presented as Mean ± SD; p value: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Dot Blot, Methylation, Expressing, Gene Expression, Quantitative Proteomics, Reverse Transcription, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Top differentially expressed lncRNAs in myoblasts and differentiated myotube.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Features of lncRNA m 6 A methylation in undifferentiated and differentiated muscle. (A) The enriched consistent motif of m 6 A peaks in lncRNAs in GM and D4. (B) Metagene profiles of enrichment of all m 6 A peaks across lncRNAs transcriptome. (C) The top: pie charts represent the proportion of m 6 A peaks in the three regions of lncRNAs at GM. The bottom: histogram represents the relative enrichment of m 6 A peaks in the three regions of lncRNAs at GM. (D) The top: pie charts represent the proportion of m 6 A peaks in the three regions of lncRNAs at D4. The bottom: histogram represents the relative enrichment of m 6 A peaks in the three regions of lncRNAs at D4. (E) The frequence of m 6 A Peak Numbers in lncRNAs in GM and D4. (F) Bar plot shows the Numbers of m 6 A methylated lncRNAs in GM and D4. Blue represents 74 hyper-methylated lncRNAs in GM, while yellow represents 83 hyper-methylated lncRNAs in D4.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Methylation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Differentially m 6 A modified lncRNAs in undifferentiated and differentiated muscle. (A) Distribution of genes with a significant change in both the m 6 A methylation and RNA expression levels before (GM) and after differentiation (D4), different colors were used to identify representative genes. And 15 m 6 A methylated significantly differently expressed lncRNAs were marked. (B) qRT-PCR validated the 15 m 6 A methylated significantly differently expressed lncRNAs in developing muscle cells. (C) Real-time PCR detection of the expression of the 15 m 6 A methylated significantly differently expressed lncRNAs in immunoprecipitated RNAs. IgG Immunoprecipitation was used as negative control. Quantitative data was represented as Mean ± SD; p value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, no significant difference. (D) Integrative Genomics Viewer (IGV) plots show the m 6 A peaks of lncRNA Xist, Ptgs2os2 and Brip1os were highly enriched in m 6 A-RIP data. (E) Real-time PCR detection of the expression of the 5 hyper-upregulated and 5 hypo-downregulated lncRNAs in immunoprecipitated RNAs of GM and D4. Data were normalized by IgG Immunoprecipitation. Quantitative data was represented as Mean ± SD; p value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, no significant difference.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Modification, Methylation, RNA Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: m 6 A methylation levels were positively correlated with the abundance of lncRNAs. (A) Scatter plot shows the positive correlation between m 6 A levels (significant changes) and expression values of lncRNAs with significantly differential expression between GM and D4, adjusted P ≤ 0.05. (B) Scatter plot shows no correlation between m 6 A levels (no significant changes) and expression values of lncRNAs without significantly differential expression between GM and D4. (C) Cumulative frequency of log2FC for lncRNAs containing m 6 A or without m 6 A methylation in GM. Kolmogorov-Smirnov test was used to estimated inter-group difference. (D) Cumulative frequency of log2FC for lncRNAs containing m 6 A or without m 6 A methylation in D4. Kolmogorov-Smirnov test was used to estimated inter-group difference.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Methylation, Expressing, Quantitative Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Myogenesis associated lncRNAs are regulated by m 6 A methyltransferase METTL3. (A) qRT-PCR shows the RNA expression level of METTL3 in METTL3-overexpressing C2C12 cells. GFP-overexpressing C2C12 cells as negative control. (B) Western blot detected the protein expression levels of METTL3 in METTL3-overexpressing C2C12 cells. (C) qRT-PCR shows the expression of myogenesis associated lncRNAs in METTL3-overexpressing C2C12 cells. (D) qRT-PCR shows the RNA expression level of METTL3 in METTL3 knockdown C2C12 stable cell lines. A nonsense sequence constructed to dCas9 repressor as negative control. (E) Western blot detected the protein expression levels of METTL3 in METTL3 knockdown C2C12 stable cell lines. (F) qRT-PCR shows the expression of myogenesis associated lncRNAs when METTL3 was knockdown. Data are presented as Mean ± SD; p value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, RNA Expression, Negative Control, Western Blot, Expressing, Knockdown, Stable Transfection, Sequencing, Construct

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization of Long Non-coding RNAs Modified by m 6 A RNA Methylation in Skeletal Myogenesis

doi: 10.3389/fcell.2021.762669

Figure Lengend Snippet: Functional relevance between m 6 A methylated lncRNAs and their adjacent mRNAs. (A) The top ten GO terms of the adjacent mRNAs (adjusted P ≤ 0.05) that related to differentially expressed lncRNAs in muscle cells. (B) The top twelve KEGG pathways of the adjacent mRNAs (adjusted P ≤ 0.05) that related to differentially expressed lncRNAs in muscle cells. (C) Table shows seven pairs of significantly differently expressed and methylated lncRNAs and their adjacent mRNAs in muscle cells. All have a significant threshold of FDR-adjusted p value ≤0.05. FDR, False Discovery Rate. (D) qRT-PCR shows the adjacent mRNA expression in GM and D4. (E) The effects of si-lncRNAs on the RNA expression levels of the corresponding lncRNA and mRNA. Data are presented as Mean ± SD; p value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions.

Techniques: Functional Assay, Methylation, Quantitative RT-PCR, Expressing, RNA Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: Interaction of MBD1 with MPG. (A) Structure of MBD1 and MPG. MBD1 (isoform v3) contains the MBD, nuclear localization signal (NLS), cysteine-rich CXXC domains, and TRD (9, 37). TRD (amino acids 473–536) was used in a yeast two-hybrid screen as a bait. MPG has an enzymatically active site at glutamic acid (E) 125. In full-length and deletion mutants of MPG, the presence and absence of MBD1 interaction in Fig. 2 A are indicated by plus and minus, respectively. (B) Association between endogenous MBD1 and MPG in HeLa cells. (C) Direct binding of the TRD of MBD1 to MPG in vitro. Bacterially expressed (His)6-TRD of MBD1 and GST-MPG were used for nickel-NTA resin affinity chromatography (38). The arrowhead indicates the full-length MPG fused to GST.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Two Hybrid Screening, Binding Assay, In Vitro, Affinity Chromatography

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: Regions of MPG for interacting with MBD1. (A) Association of MPG with the TRD of MBD1. GST-fused TRD of MBD1 or GST was immobilized on glutathione agarose beads and incubated with the lysates from HeLa cells expressing full-length or deletion mutants of MPG. (B) Localization of MPG and MBD1 in the nucleus. The full-length or deletion mutants of FLAG-MPG were coexpressed with DsRed-fused MBD1.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Incubation, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: Cooperation of MBD1 and MPG for transcriptional repression. GAL4-MPG and FLAG-MBD1 (A), GAL4-TRD of MBD1 and FLAG-MPG (B), and GAL4-TRD of MBD1 and FLAG-MPG mutants (C) were expressed in HeLa cells to examine their effects on a luciferase reporter that contains five GAL4-binding elements just upstream of human SNRPN or the p16 gene promoter. The cells were transfected with plasmids (1 μg) to express FLAG-tagged proteins, together with indicated amounts of plasmids for GAL4-fused proteins. FLAG-mock was used as a control. The luciferase activities from insertless GAL4-mock were normalized to 100. Values are given as means and standard deviations of results from more than three independent experiments.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Luciferase, Binding Assay, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: Molecular dynamics of MBD1 and MPG under MMS-induced DNA damage. (A) MMS-dependent growth inhibition of NCI-H1299 and SBC-5 cells. Cell numbers were examined after MMS treatment for 1 h and after incubation for 3 days. (B) Expression of MBD1, MPG, and β-tubulin in untreated and MMS-treated cells. At 48 h after transfection, both cell lines expressing HA-MBD1 and FLAG-MPG were treated with 1 mM MMS for 1 h. Western blot analysis was done with anti-MBD1, anti-FLAG, and anti-β-tubulin antibodies. (C) Association of MBD1 and MPG with chromosomal p16 gene promoters under MMS treatment. The p16 gene promoter is highly methylated in NCI-H1299 cells and unmethylated in SBC-5 cells (36, 37). For chromatin immunoprecipitation, the cells expressing HA-MBD1 and FLAG-MPG were treated with 1 mM MMS for 1 h and incubated for the indicated times. The coprecipitated DNAs with the indicated antibodies were PCR-amplified by using a set of primers for p16 promoter sequences. Genomic DNAs in the input cell lysates before the immunoprecipitation were used as a control. All data are from more than three independent experiments.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Inhibition, Incubation, Expressing, Transfection, Western Blot, Methylation, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: Abrogation of methylated DNA binding of MBD by the presence of 7-mG in a methyl–CpG pair. (A) Construction of 30 base-paired oligodeoxynucleotides containing 5-mC and/or 7-mG. The unique dinucleotides, CG × CG, mCG × mCG, mCG × mCmG, and CG × CmG, were introduced into position (XG × XY). mG or G was incorporated into position Y of the synthesized strand. (B) Electrophoretic mobility-shift assay by using the oligodeoxynucleotides and MBD from MBD1. (C) Structural model of the interaction between MBD and methylated DNA. A proposed interaction between the methyl–CpG site and two arginine side chains at the interface, on the basis of NMR structure determination, is shown in A. The interactions between the guanine bases and the arginine side chains (b and d) can be perturbed by alkylations at N7 positions of the guanine bases (c and e). Arginine 22 and 44, blue; DNA, red; hydrogen bond between arginine and guanine, white; methyl groups in methyl–CpG sequence, yellow.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Methylation, Binding Assay, Synthesized, Electrophoretic Mobility Shift Assay, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

doi: 10.1073/pnas.2131819100

Figure Lengend Snippet: MBD1 knockdown increases cell sensitivity to MMS. (A) Effectiveness of siRNA knockdown of MBD1. The cells were transfected with siRNAs targeting mRNAs encoding MBD1 and lamin A/C. MPG and β-tubulin are also detected. (B) Effect of MBD1 knockdown on cell growth inhibition by MMS. Growth inhibition assay was performed as in Fig. 4A. GL3 siRNA was used as a control (41). Values are given as means and standard deviations from five independent experiments.

Article Snippet: Small Interfering RNA (siRNA) Knockdown of MBD1. siRNA duplexes were designed for targeting mRNA encoding human MBD1 (Japan Bioservice, Saitama, Japan): 5′-GGCAUCUUGUGCUAUCCAGTT-3′ and 5′-CUGGAUAGCACAAGAUGCCTT-3′.

Techniques: Transfection, Inhibition, Growth Inhibition Assay

Journal: Cancers

Article Title: Netupitant Inhibits the Proliferation of Breast Cancer Cells by Targeting AGK

doi: 10.3390/cancers16223807

Figure Lengend Snippet: Netupitant demonstrates the potential to inhibit the proliferation of breast cancer cells. ( A ) Validation of gene silencing efficiency for three siRNA sequences. ( B ) Growth curve assay following AGK gene silencing. ( C ) Clonal formation assay subsequent to AGK gene silencing. ( D ) Identification of five candidate compounds targeting AGK through virtual screening. ( E ) Assessment of the inhibitory capacity of these five candidate compounds on breast cancer cell proliferation via CCK8 assay. ( F ) Root Mean Square Deviation (RMSD) analysis of AGK and Netupitant obtained from molecular dynamics simulation. ( G ) Binding site analysis showing where Netupitant interacts with AGK, including the relevant amino acids involved in this interaction. ( H ) BIL analysis conducted to evaluate the affinity between AGK protein and Netupitant. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After a 24 h incubation, each well was treated with a mixture consisting of 5 μL of siRNA designed to target AGK (RiboBio, Guangzhou, China) and 5 μL of RNAiMAX (Invitrogen, Carlsbad, CA, USA).

Techniques: Biomarker Discovery, Tube Formation Assay, CCK-8 Assay, Binding Assay