Journal: Cell stem cell

Article Title: GFAP mutations in astrocytes impair oligodendrocyte progenitor proliferation and myelination in a human iPSC model of Alexander disease

doi: 10.1016/j.stem.2018.07.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The pHIV-hGFAP-GFP lentiviral vector was prepared by cloning the hGFAP-GFP fragment from the hGFAP-GFP vector (Addgene, plasmid #40592) into the pHIV vector.

Techniques: Recombinant, SYBR Green Assay, Software

Journal: Nature

Article Title: Mechanisms of action and resistance in histone methylation-targeted therapy

doi: 10.1038/s41586-024-07103-x

Figure Lengend Snippet: a , Characteristics of clinically resistant clones based on single-cell analysis and genomic profiling. b , Chronological transition of normalized VAF values for somatic mutations in Pt2 in relation to treatment with valemetostat. c , tSNE projection for Pt2 scRNA-seq data. DNMT3A expression was upregulated in the recurrent clone ( P < 10 −9 ). d , scATAC-seq and ChIP-seq tracks at DNMT3A locus. e , Venn diagram depicts overlapped chromatin-condensed inactivated genes (Promoter sum <0.01) in Pt2 tumor cells at Pre, CR, and PD ( DNMT3A expression). f , Scatter plots show all gene promoter activities in recurrent clones (y-axis) in Pt2 (left) and Pt3 (right) versus corresponding normal cells (x-axis). The gene loci with decreased copy numbers as defined from the WGS data are indicated in dark purple. g , Histogram shows differentially methylated (ΔmCpG <−10%, or >10%) probes in Pt2 resistant tumor at PD (135 weeks) versus pre-treatment tumor. h , i , Boxplots summarize normalized log 2 fold changes of scATAC-seq promoter activities ( h ) and scRNA-seq gene expression ( i ) in relation to treatment-associated mCpG gain in Pt2. The genes for which integrated data were available were evaluated. Statistical significance is provided only for main combinations. j , Normalized log 2 fold changes of scATAC-seq promoter activities of tumor suppressor genes (TSG) in relation to treatment-associated mCpG gain in Pt2. The genes for which integrated data were available were evaluated. Statistical significance is provided only for main combinations. k , Pt2-derived resistant cell line was successfully established. The same clonal origin and the absence of PRC2 mutations were confirmed by targeted sequencing. Bar graph shows relative RNA levels of DNMT family and PRC2 genes. l , Table summarizes characteristics of ATL cell lines. Pt2_PD cells showed low sensitivity to valemetostat. m , Knockdown and DNMT family and PRC2 genes were induced by lentivirus-mediated shRNA. After lentivirus infection, growth cell numbers for 6 days were calculated. The middle lines within box plots correspond to the medians; lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR. Statistics and reproducibility are described in Methods.

Article Snippet: For stable expression of DNMT in lymphoma cells, haemagglutinin-tagged DNMT3A and DNMT3B cDNA were subcloned into the lentivirus vector pHIV-dTomato (Addgene #21374).

Techniques: Clone Assay, Single-cell Analysis, Expressing, ChIP-sequencing, Methylation, Gene Expression, Derivative Assay, Sequencing, Knockdown, shRNA, Infection, Whisker Assay

Journal: Nature

Article Title: Mechanisms of action and resistance in histone methylation-targeted therapy

doi: 10.1038/s41586-024-07103-x

Figure Lengend Snippet: a , ATL cell lines were cultured in growth media supplemented with 10 nM of valemetostat for two months. Inhibitor-resistant outgrowth was observed at 100 nM. Bar graph shows VAF values of TET2 W1847X DNA and expressed mRNA in ATN-1 parental and valemetostat-resistant cells. b , shRNA targeting EZH1, EZH2, or EED were introduced by lentivirus vectors in parental and resistant cell line (ATN-1_R). Graphs show cell growth (%) relative to control shRNA. c , Growth inhibition rate (%) by valemetostat (0 ~ 10,000 nM) in ATN-1 parental and valemetostat-resistant cells. n = 3 independent experiments, mean ± SD. For gene rescue experiment, TET2 cDNA were transduced by lentivirus vector. d , Histograms show differentially methylated (ΔmCpG <−10%, or > 10%) probes in resistant ATN-1 versus parental cells. e , f , H3K27me3 occupancy was analyzed by ChIP-seq for the parental and valemetostat-resistant ATN-1 cells. Boxplot shows H3K27me3 log 10 signals in relation to resistance-associated mCpG gain ( e ). The genes for which integrated data were available were evaluated. Statistical significance is provided only for main combinations. Representative tracks for H3K27me3 and methylated CpG tracks are shown in ( f ). Arrowheads indicate representative CpG sites with methylation gain. g , Log 2 fold-changes of RNA-seq expression level (TPM) at mCpG gain genes (mCpG UP sites > 2) in resistant ATN-1 cells compared to parental cells. h , MSP was performed for DNA isolated from parental or resistant ATN-1 cells in the presence or absence of TET2 . Amplified DNA was visualized by agarose gel electrophoresis for TSG loci with primer sets specific for methylated state (M) or unmethylated state (U). Data are representative of two independent experiments. NTC: no template control. i , Heatmaps represent recovered outgrowth cell numbers in ATN-1 cells expressing shTET2 (#1, #2) in 96-well plate culture. Collected outgrowth clones ( n = 16) are indicated. j , TET2 RNA level in randomly collected outgrowth clones quantified by qRT-PCR. k , H3K27me3 level in valemetostat outgrowth clones. l , Scatter plot shows DNA methylation changes (x-axis) and accumulation of H3K27me3 (y-axis) in the promoter proximal region (TSS ± 1 kbp) of each gene in the outgrowth shTET2 clone #1. Values are averaged per gene and represented only differentially methylated genes (ΔmCpG < −5% or >5%). m , MSP assay for H3K27me3 target genes ( CDKN1A and BCL2L11 ) in valemetostat outgrowth clones. n , Bar graphs show differentially methylated CpG sites in shTET2 outgrowth clone (left) and Pt3 PD clone (right) in single nucleotide resolution analysis using EM-seq data. Percentages were compiled from all CpG sites (filter depth > 5) in the TSS and downstream gene body regions (center ± 1 kbp). Target genes were defined based on H3K27me3, SUZ12, and H3K27ac ChIP-seq data. o , Pie chart shows the percentage of epigenomic domains of CpG islands near the TSS with increased methylation ( P < 0.05). p , q , Control cells (shCtrl) and the recovered outgrowth clones were treated with valemetostat (100 nM) and DAC (10 nM). Bar graphs show relative cell growth at 14 days ( p , n = 3, independent experiments, mean ± SD) and relative expression levels of the H3K27me3 target genes ( CDKN1A and BCL2L11 ) at 7 days ( q ). The middle lines within box plots correspond to the medians; lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR. Statistics and reproducibility are described in Methods. For gel source data, see Supplementary Fig. .

Article Snippet: For stable expression of DNMT in lymphoma cells, haemagglutinin-tagged DNMT3A and DNMT3B cDNA were subcloned into the lentivirus vector pHIV-dTomato (Addgene #21374).

Techniques: Cell Culture, shRNA, Control, Inhibition, Plasmid Preparation, Methylation, ChIP-sequencing, RNA Sequencing, Expressing, Isolation, Amplification, Agarose Gel Electrophoresis, Clone Assay, Quantitative RT-PCR, DNA Methylation Assay, MSP Assay, Whisker Assay

Journal: Nature

Article Title: Mechanisms of action and resistance in histone methylation-targeted therapy

doi: 10.1038/s41586-024-07103-x

Figure Lengend Snippet: a , Relative expression levels of DNMT family genes in TL-Om1 parental and valemetostat-resistant cells quantified by qRT-PCR. b , shRNA targeting EZH1, EZH2, or EED were introduced by lentivirus vectors in parental and resistant cell line (TL-Om1_R). Graphs show cell growth (%) relative to control shRNA. c , Growth inhibition rate (%) by valemetostat (0 ~ 10,000 nM) in TL-Om1 parental and valemetostat-resistant cells. shRNA targeting DNMT3A or DNMT3B were transduced by lentivirus vector. n = 3, independent experiments, mean ± SD. d , Histograms show differentially methylated (ΔmCpG <−10%, or > 10%) probes in resistant TL-Om1 versus parental cells. e , MSP assay in TL-Om1_R with shDNMT3A. Amplified DNA was visualized by agarose gel electrophoresis for TSG loci with primer sets specific for methylated state (M) or unmethylated state (U). Data are representative of two independent experiments. f , DNMT3A (WT and E629stop which lacks enzymatic domain) and DNMT3B expressing cell models were established by lentivirus vectors in ATL-derived TL-Om1 and ATN-1 cells and DLBCL-derived WSU-DLCL2 cells. DNMT3A and DNMT3B protein levels were analyzed by immunoblotting (ATN-1). Data are representative of two independent experiments. g , Cell growth curves show recovered outgrowth cell numbers in ATN-1 cells in 96-well plate culture. h , DNMT3A RNA level in randomly collected outgrowth clones ( n = 16) quantified by qRT-PCR. i , Scatter plot shows DNA methylation changes (x-axis) and accumulation of H3K27me3 (y-axis) in the promoter proximal region (TSS ± 1 kbp) of each gene in the outgrowth DNMT3A clone #1. Values are averaged per gene and represented only differentially methylated genes (ΔmCpG < −5% or >5%). j , Bar graphs show differentially methylated CpG sites in DNMT3A outgrowth clone (left) and Pt5 PD clone (right) in single nucleotide resolution analysis using EM-seq data. Percentages were compiled from all CpG sites (filter depth > 5) in the TSS and downstream gene body regions (center ± 1 kbp). Target genes were defined based on H3K27me3, SUZ12, and H3K27ac ChIP-seq data. k , Pie chart shows the percentage of epigenomic domains of CpG islands near the TSS with increased methylation ( P < 0.05). l , m , Parental cells and the recovered outgrowth clones were treated with valemetostat (100 nM) and DAC (10 nM). Bar graphs show relative cell growth at 14 days ( l , n = 3, independent experiments, mean ± SD) and relative expression levels of the H3K27me3 target genes ( CDKN1A and BCL2L11 ) at 7 days ( m ). Statistics and reproducibility are described in Methods. For gel source data, see Supplementary Fig. .

Article Snippet: For stable expression of DNMT in lymphoma cells, haemagglutinin-tagged DNMT3A and DNMT3B cDNA were subcloned into the lentivirus vector pHIV-dTomato (Addgene #21374).

Techniques: Expressing, Quantitative RT-PCR, shRNA, Control, Inhibition, Plasmid Preparation, Methylation, MSP Assay, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Western Blot, Clone Assay, DNA Methylation Assay, ChIP-sequencing

Journal: Stem Cell Research & Therapy

Article Title: Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector

doi: 10.1186/s13287-019-1416-5

Figure Lengend Snippet: Robust, coordinated induction of HNF4A, HNF1A, FOXA3, and GFP transgenes. a Scheme of the DOX-inducible lentiviral vector TetO-HHFG, containing three different 2A peptides. b Schematic representation of the reprogramming strategies with and without the inducible reprogramming vector, TetO-HHFG (see “ ” for details). c Early time response of DOX-dependent induction of HNF4A, HNF1A, FOXA3, and GFP mRNA, measured by qRT-PCR in HDF-LT DOX treated with 1000 ng/mL DOX (DOX). Values are referred to untreated HDF-LT DOX ( T = 0; noDOX). Regression table is on the right. Data shown is represented as mean ± s.d. from two experiments with three biological replicates. d Immunofluorescence of HNF4A, HNF1A, FOXA3, and GFP in HDF-LT DOX treated without (noDOX) or with 1000 ng/mL DOX (DOX) for 3 days. Bar equals 50 μm. Quantification of immunofluorescence results is shown on the right

Article Snippet: The lentiviral vector pHIV-EGFP-FOXA3 was obtained by PCR-cloning human FOXA3 cDNA into BamHI-XbaI restriction sites in pHIV-EGFP [ ] (Addgene #21373).

Techniques: Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector

doi: 10.1186/s13287-019-1416-5

Figure Lengend Snippet: Phenotype reversal of iHEP-LT DOX . a Schematic representation of time-course collection of reprogramming intermediates. Reprogramming of HDF-LT DOX was initiated at day 0 by addition of DOX 250 ng/mL. The collection point labeled 10 +d represents cells reprogrammed for 10 days in DOX followed by specified days (in superscript) without DOX. b , c Expression of exogenous and endogenous reprogramming factors across a 0–17-day timeline. d , e Expression of exogenous transgenes and selected hepatic mRNA during the timeline described in a . mRNA levels were quantified by qRT-PCR and expressed relative to the expression at 10 days. f Scheme of the bicistronic lentiviral vectors independently expressing HNF4A, HNF1A, and FOXA3 together with mutually compatible fluorescent proteins. g HDF-LT cells were co-infected with HNF4A, HNF1A, and FOXA3 lentiviral vectors and cultured in HMM media. Expression of fluorescent proteins was assessed by flow cytometry after 30 days

Article Snippet: The lentiviral vector pHIV-EGFP-FOXA3 was obtained by PCR-cloning human FOXA3 cDNA into BamHI-XbaI restriction sites in pHIV-EGFP [ ] (Addgene #21373).

Techniques: Labeling, Expressing, Quantitative RT-PCR, Infection, Cell Culture, Flow Cytometry

Journal: Cancer Science

Article Title: MicroRNA‐93 targets WASF3 and functions as a metastasis suppressor in breast cancer

doi: 10.1111/cas.14423

Figure Lengend Snippet: MicroRNA (miR)‐93 suppresses 3D organoid formation capacities. A, 3D organoid‐forming capacity of breast cancer cells (MDA‐MB‐231 and MCF‐7). Cells were infected with a lentivirus vector driving constitutive expression of miR‐93 or its inhibitor. n = 3, * P < .05. Scale bar = 100 μm. B, Expression levels of genes associated with cancer stem cell properties and epithelial‐mesenchymal transition in breast cancer cells forming organoids. C, cells of control organoid; m93, cells of miR‐93‐expressing organoid. Data are presented as mean ± SD. n = 3, * P < .05

Article Snippet: The sequence of precursor miR‐93 (mature miR‐93 and its 5′‐ and 3′‐ flanking regions) and the full‐length coding region of the WASF3 mRNA (NM_006646.6 [GenBank]) were amplified by PCR and cloned into the pEIZ‐HIV‐ZsGreen or mCherry lentivirus vector (Addgene: #18121) or the pLentiLox3.7‐EF1α‐mCherry vector, a derivative of pLentiLox3.7 (Addgene: #11795), respectively.

Techniques: Infection, Plasmid Preparation, Expressing, Control

Journal: Cancer Science

Article Title: MicroRNA‐93 targets WASF3 and functions as a metastasis suppressor in breast cancer

doi: 10.1111/cas.14423

Figure Lengend Snippet: MicroRNA (miR)‐93 suppressed liver metastasis in vivo. A, Schematic representation of the splenic injection of tumor cells and competitive transplantation assays. For the competitive transplantation assay, MDA‐MB‐231‐ZsGreen cells overexpressing miR‐93 (miR‐93 ZsGreen) and MDA‐MB‐231 mCherry competitor (mixed 1:1) were transplanted into the spleen of immunodeficient NSG mice. B, Gross examination of the development of metastases in the liver 21 d after intrasplenic injection of MDA‐MB‐231 cells stably infected with miR‐93‐expression or control lentivirus. Number of tumors on the surfaces of the liver was counted. Arrows indicate representative liver metastases. Data are presented as mean ± SD. n = 6, * P < .05. Scale bar = 10 mm. C, Donor chimerism of the cancer cells metastasized to the liver was evaluated by FACS. The liver was perfused, dissociated, and analyzed 3 wk after transplantation. Percentage of donor chimerism of the cancer cells metastasized in the liver after competitive transplantation assay. Data are presented as mean ± SD. n = 3, * P < .05

Article Snippet: The sequence of precursor miR‐93 (mature miR‐93 and its 5′‐ and 3′‐ flanking regions) and the full‐length coding region of the WASF3 mRNA (NM_006646.6 [GenBank]) were amplified by PCR and cloned into the pEIZ‐HIV‐ZsGreen or mCherry lentivirus vector (Addgene: #18121) or the pLentiLox3.7‐EF1α‐mCherry vector, a derivative of pLentiLox3.7 (Addgene: #11795), respectively.

Techniques: In Vivo, Injection, Transplantation Assay, Stable Transfection, Infection, Expressing, Control