Journal: Coronaviruses

Article Title: Transcriptome Analysis of Feline Infectious Peritonitis Virus Infection

doi: 10.1007/978-1-4939-2438-7_20

Figure Lengend Snippet: Work flow and settings for CLC bio GWB software analysis

Article Snippet: Fig. 2 Work flow and settings for CLC bio GWB software analysis Conduct RNA-seq analysis with the settings stated in the Fig. , using trimmed raw sequences.

Techniques: Software

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Left, Generic structure of bEXOmiRs before and after processing. bEXOmiRs contain a random 15 nucleotide (nt) (base-paired) sequence followed by a constant exosome-targeting motif (GGAGGAG, ‘EXO motif’). Middle, intracellular trafficking of bEXOmiRs before export via EVs (exosomes and/or microvesicles). Once secreted, bEXOmiRs can be detected in purified EV fractions by RT-PCR or next generation sequencing. ( B ) Three example bEXOmiRs ( A, B, C ; ) were cloned into a mammalian expression vector and transfected into HEK293T cells. bEXOmiR expression in both EV (upper panel) and cellular (bottom panel) fractions was assessed by Stem-Loop RT-PCR. Negative control RT-PCR devoid of RT-primer is shown in lane ∅. Red asterisks indicate non-specific PCR amplification bands. ( C ) HEK293T cells transfected with control or RAB27A siRNA. Fresh media was added 24 hr post-transfection; EVs were then isolated 48 hr later. Semi-quantitative RT-PCR gel images (top) show relative levels of a bEXOmiR reporter in both cellular (Cells) and EV fractions from control and RAB27A siRNA-treated cells from two experiments. Graph shows bEXOmiR level after RAB27A depletion, as quantified by qRT-PCR (n = two independent experiments, EXP#1 and EXP#2). Immunoblots at bottom show depletion of RAB27A; α-Tubulin was a loading control. Marker mobility is shown at left in basepairs ( B and C ) top panels) or kilodaltons ( C bottom panels). ( D ) Reproducibility of 5000 bEXOmiR abundances measured by deep sequencing in isolated EVs from two replicate cultures (REP1 and REP2) of K562 cells previously infected with the 5,000-bEXOmiR test library (see text).

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Sequencing, Purification, Reverse Transcription Polymerase Chain Reaction, Next-Generation Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Negative Control, Amplification, Control, Isolation, Quantitative RT-PCR, Western Blot, Marker, Infection

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Detailed design of bEXOmiR reporters. The double stranded stem-region contains a 15 nt-random sequence (barcode; highlighted in blue) with perfect base complementarity. Three examples of barcode sequences, A, B and C are shown at right. The barcode sequence is then followed by a constant 7nt exosome-targeting motif (EXO motif, green) that contains two asymmetric bulges (one in each strand; a, bottom). The EXO motif is then followed by a loop sequence (orange); both were derived from endogenous hsa-miR-601. Finally, mini-miR-30 context sequences were placed at the base of the stem region (red). N indicates any ribonucleotide base. ( B ) Left, two protocols (P1 and P2) for EV isolation used in this study. Biochemical characterization of whole cell lysate (Cells) and EV fractions derived from P1 and P2 is shown in immunoblot panels at right using antibodies for LAMP1, CD81, Calnexin, Golgin 97, α-Tubulin and GDI-β. Molecular mass marker mobility is shown at left in kilodaltons. ( C ), Schematic for Stem-loop RT-PCR protocol used to detect bEXOmiRs in extracted RNA samples. Panel at right, lane 1 (EVs) shows expression of bEXOmiR-C (panel A ) detected by RT-PCR using RNA extracted from EVs released during 48 hr from HEK293T cells transfected with a bEXOmiR-expression vector. Reactions lacking either Stem-Loop primer (ΔFS-primer control, Lane 2) or reverse transcriptase (RT (-), Lane 3) are shown. Molecular marker mobility is shown at left in base pairs. ( D ) bEXOmiR-expressing HEK293T cells were grown for 48 hr prior to EV isolation and flotation using a discontinuous sucrose gradient; the presence of the bEXOmiR reporter was determined by RT-PCR at the indicated gradient interface (dotted red arrow), as shown in lane 1 (floated EVs) of the agarose gel at right. No bEXOmiR signal was observed in reactions lacking Stem-loop primer (lane 2). ( E ) RNase protection assay. Isolated EVs from bEXOmiR-expressing K562 cells were incubated with PBS (gray bar), PBS + RNase (black bar) or PBS + TX100 + RNase (red bar). t test: *p<0.05; error bars represent SEM; n = 3. ( F ) and ( G ), bEXOmiR-expressing K562 cells were treated with 10 nM Bafilomycin A1 (BA1) for 20 hr before EV isolation. Immunoblot analysis of control (DMSO) and BA1-treated (BA1) cells and EVs is shown in ( F ). RT-PCR detection of bEXOmiR reporter in cells and EV fractions from control and BA1-treated cells is shown in ( G ). ( H ) Schematic representation of the design and composition of the initial bEXOmiR test library.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Sequencing, Derivative Assay, Isolation, Western Blot, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Agarose Gel Electrophoresis, Rnase Protection Assay, Incubation

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: Top, K562 cells visualized after centrifugation onto coverslips, immunostained for CD63; bottom, quantitation of total CD63-vesicle area. Each dot represents a single, CD63-positive structure in 18 or 20 parental or bEXOmiR expressing cells, respectively (n ~ 600 structures counted for each condition; mean ~32 structures per cell for the parental and ~34 for the bEXOmiR expressing cells. Red line indicates the median area for each population.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Centrifugation, Quantitation Assay, Expressing

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Diagram depicting key steps of the screen performed in this study. Oligonucleotides encoding sgRNA-bEXOmiR pairs were designed computationally and then synthesized using solid-phase technology. Next, oligonucleotides were pool-cloned (1) into a Lentivirus vector that drives expression of both sgRNA and bEXOmiR under U6 and EF1α promoters, respectively. WT and Cas9-positive K562 cells were then infected at low MOI (2), such that after infection and selection, each cell expresses only a single sgRNA-bEXOmiR pair (3). This approach enables identification and quantification of both EVs and their respective cell of origin via unique molecular identifiers: barcodes and sgRNAs respectively. sgRNA-bEXOmiR-expressing cells were then grown for 48 hr before collection of culture supernatants from which EVs were purified (4). Finally, barcode abundance was measured, comparing WT and Cas9-expressing cells, by deep sequencing from EV-extracted RNA . ( B ), Initial pilot screen using a 25,000 bEXOmiR-sgRNA library targeting membrane trafficking, mitochondrial and motility (MMM) genes. Colored insets display zoomed activator (red nodes in red inset) and suppressor (blue nodes in blue inset) hits that passed effect size cutoffs (≤ −2 or ≥+2) and had a -Log p-value>1.5. ( C ), genome-wide (minus MMM sublibrary) screen. Colored insets display zoomed activator and suppressor hits that passed effect size cutoffs (≤ −2 or ≥+2) and had a -Log p-value>2.5. Green labels in ( B ) and ( C ) indicate previously known EV regulators; yellow labels represent genes that regulate miRNA trafficking or processing.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Synthesized, Clone Assay, Plasmid Preparation, Expressing, Infection, Selection, Purification, Sequencing, Membrane, Genome Wide

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ), Quantitative Nanostring miRNA profiling of both cellular (Cells) and EV fractions was performed to compare endogenous miRNA signatures in SMPD3 CRISPR knockout (KO) or ARHGEF18 KO K562 cell lines versus control cells; blue and green violin plots, respectively. miRNA abundance is plotted as percent of miRNA abundance in control cells. Violin plots show overall distribution of common EV-associated miRNAs (circles) detected in two independent experiments. Three specific endogenous miRNAs, hsa-miR-19a-3p (black), hsa-miR-151a-3p (red) and hsa-miR-548b-3p (blue) are indicated. ( B ) and ( C ), Biochemical analysis of EV fractions in CRISPR knockout cell lines from selected hits derived from the initial MMM-focused ( B ) or from the rest of the genome-wide ( C ) screen. EV extracts were immunoblotted with anti-HSC70, anti-Flotillin or anti-CD81 antibodies; numbers at left indicate mobility of molecular weight markers in kD. Bars indicate standard deviation from two independent sets of two replicate experiments. ( D ) Fusion of pHluorin-CD63-positive structures at the surface of HeLa cells observed using TIRF microscopy of fluorescent siRNA-transfected cells. Each dot represents number of events per cell over 90 s. ( E ) and ( F ), Characterization of purified EVs by Nanoparticle Tracking Analysis (NTA). ( E ) Representative plot of EV size distribution. Black line and red-shaded areas indicate mean value and SEM of particle concentration at each diameter, respectively, determined after three measurements. ( F ) Yield comparison of EVs derived from control and CRISPR knockout (KO) cells.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: CRISPR, Knock-Out, Control, Derivative Assay, Genome Wide, Molecular Weight, Standard Deviation, Microscopy, Transfection, Purification, Concentration Assay, Comparison

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: Activators and suppressors are labeled in red and blue letters, respectively. Inset in ( B ), ARHGEF18 depletion monitored in Cas9-expressing K562 cells by immunoblot; anti-HA was used to detect HA-tagged Cas9 expressed in this cell line, and also as a loading control. ( I ), Immunoblot validation of RAB27 sgRNAs; sgRNA #1 was used for hit validation. Numbers next to gene names refer to sgRNA reference number (10 sgRNAs per gene) used in the screen (see Materials and methods). ( J ), qPCR analysis of two endogenous miRNAs from the Nanostring analysis, to quantify the impact of loss of the indicated hits. ( K ), ARHGEF18 depletion using a different sgRNA than that used in B, or an siRNA for the NTA and live cell analysis; ( L ), mRNA levels of siRNA depleted cells used for live cell imaging in .

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Labeling, Expressing, Western Blot, Control, Biomarker Discovery, Cell Analysis, Live Cell Imaging

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Serum-starved K562 cells were treated with PBS (control) or Wnt3a (100 ng/ml) or Wnt3a + Dickkopf peptide (DKK1, 200 ng/ml) for 24 hr prior to EV isolation. Biochemical analysis of cellular (Cells) and EV fractions was performed by immunoblotting of extracts using antibodies against antigens shown. Detection of ß-Catenin was carried out using cytosolic fractions. ( B ) Quantitation of EV marker protein levels from EV immunoblots shown in ( A ). ( C ), Serum-starved K562 cells were treated with DMSO (control) or CHIR99021 (10 µM) for 24 hr prior to EV isolation. Biochemical analysis of EV fractions was performed as in ( A ). Representative immunoblots for a control experiment along with two replicate CHIR99021-treated samples are shown. ( D ) Quantitation of protein levels from EV immunoblots in ( C ). ( E ) Quantitation of LAMP1 protein from cellular fractions in ( C ). Molecular mass marker mobility is shown at the left of immunoblot panels in kilodaltons. t test: *p<0.05; **p<0.01; error bars represent SEM; n ≥ 3. ( F ) Flow cytometry determination of LAMP1 levels in anti-LAMP antibody-labeled control and CHIR99021-treated cells. Confocal images at right show lysosomes stained with anti-LAMP1 antibody (green) and nuclei labeled with DAPI (blue). Bar,10 µm.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Control, Isolation, Western Blot, Quantitation Assay, Marker, Flow Cytometry, Labeling, Staining

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Serum-starved HEK293T cells were treated with DMSO (control) or CHIR99021 (10 µM) for 24 hr before EV isolation. Representative immunoblots for a control experiment along with two replicate CHIR99021-treated samples are shown. Plot below shows quantitation of the EV fraction immunoblots above. Molecular mass marker mobility is shown at left in kilodaltons. Error bars represent standard deviation; n = 2 independent experiments. ( B ) Determination of β-Catenin stabilization upon LiCl treatment. K562 cells were treated with 10 mM NaCl (control) or increasing LiCl concentrations as indicated for 24 hr. Immunoblot analysis of cytosolic fractions was then performed with anti-β-Catenin and anti-α-Tubulin antibodies. ( C ) Cellular and EV fractions from K562 cells treated with either 10 mM NaCl or LiCl for 20 hr were analyzed by immunoblotting as indicated. Plot below shows quantitation of EV fraction immunoblots in ( C ). Molecular mass marker mobility is shown at left in kilodaltons. t test: *p<0.05; **p<0.01; error bars represent SEM; n = 3. ( D ) Serum starved RPE cells analyzed by immunoblot of the indicated proteins in EV or cellular fractions after 20 hr CHIR99021 treatment. The asterisk indicates a nonspecific band that was used as loading control.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Control, Isolation, Western Blot, Quantitation Assay, Marker, Standard Deviation

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) qPCR analysis of indicated mRNA levels in K562 cells treated for 20 hr with CHIR99021. Shown is the combined data from four independent experiments; error bars represent SEM ( t test: *p<0.05; **p<0.01). ( B ) Immunoblot of RAB27 levels in duplicate samples treated with CHIR99021 as in A. ( C ) Schematic representation of the pathway by which Wnt regulates exosome release. The link between RAB27 and GSK3 is not yet known.

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Western Blot

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , K562 , ATCC , RRID: CVCL_0004 , .

Techniques: Recombinant, Software

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: Identification of the transcription start sites of mouse Dnmt1. (A) Nucleotide sequence of the 5′-flanking region and the transcription start site of the mouse Dnmt1 showing the 299 bases upstream from the translation start codon. Boxes A–D indicate putative E2F binding sites. The transcription start site is indicated with an arrow. Exon 1o, oocyte-specific first exon; Exon 1s, somatic first exon; Inr, initiator consensus sequence (34,35). (B) Probes used in RNase protection assay. (C) RNase protection assay. RNA was prepared from NIH 3T3 cells. The major 190 base fragment protected by probe 380 is indicated with an arrow. RMW, size standards with sizes (base pair) corrected for the slower migration of double-stranded RNA based on the migration of the 80 bp double-stranded RNA protected by probe 80 (arrowhead). (D) Northern blot analysis confirmed the transcription of the 5′ region of mouse Dnmt1. Total RNA from NIH 3T3 cells was hybridized to probe 1, located 610 bases upstream from the initiator sequence, or probe 2, which corresponds to the Dnmt1 catalytic domain (33).

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Sequencing, Binding Assay, Rnase Protection Assay, Migration, Northern Blot

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: Expression of Dnmt1 mRNA during the cell cycle and in oncogenic transformed cells. (A) Northern blot showing expression of Dnmt1, cyclin D1 and cyclin E during the cell cycle. Quiescent NIH 3T3 cells were stimulated by replacement with complete medium including 10% fetal bovine serum and harvested at the indicated times. (B) Percentage of cells in each stage of the cell cycle at various times after serum stimulation. DNA amounts were measured by propidium iodide (PI) staining and FACScan analysis. The cell cycle distribution is indicated on the right of the histogram. (C) Northern blot showing expression of Dnmt1 in transformed 3Y1 cells, with gapdh as an internal control. WT, parental 3Y1 cells; SR, RSV-transformed 3Y1 cells; HR, Ha-ras transformed 3Y1 cells; E1A, E1A-transformed cells; SV, SV40-transformed 3Y1 cells.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Expressing, Transformation Assay, Northern Blot, Staining

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: Identification of the core promoter region of mouse Dnmt1 and two critical binding sites. (A) COS-7 cells were transfected with several promoter fragments linked to a luciferase reporter construct. Results are shown as relative luciferase activity after 48 h. Bars indicate standard deviations (SD). COS-7 cells were transfected with serial deletion constructs of the upstream region: 2061-luc, 736-luc, 500-luc, 300-luc, 100-luc, R736-luc (the 736 bp upstream region in reverse orientation) and No insert-luc (PGV-B2) constructs. (B) COS-7 cells were transiently transfected with constructs containing the first 300 bases upstream of the start codon either with wild-type or mutated (crosses) putative E2F binding sites, or with No insert-luc (PGV-B2). Luciferase activity of the wild-type 300-luc (column 1) was defined to be 100%. (C) As in (B), except that NIH 3T3 cells were used instead of COS-7 cells.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Binding Assay, Transfection, Luciferase, Construct, Activity Assay

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: Promoter activity of the core promoter region of mouse Dnmt1 in transformed 3Y1 cells. (A) Several cell lines were transiently transfected with the 300-luc construct and luciferase activity was measured as in Figure ​Figure3.3. WT, parental 3Y1 cells; SR, RSV-transformed 3Y1 cells; HR, Ha-ras transformed 3Y1 cells; E1A, E1A-transformed cells; SV, SV40-transformed 3Y1 cells. (B) SV-3Y1 cells were transfected with the 300-luc construct, a construct with mutated site A (300 A), a construct with mutated site C (300 C) or a construct in which both sites A and C were mutated (300 AC). Numbers shown are fold induction relative to luciferase activity of 300-luc. (C) Comparison of the core promoter region of Dnmt1 between mouse and rat. Inr, initiator sequence: sites A and C correspond to those shown in Figure ​Figure22A.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Activity Assay, Transformation Assay, Transfection, Construct, Luciferase, Sequencing

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: EMSAs to identify molecules bound to site A and site C in the core promoter region of mouse Dnmt1. (A) Complex formation of 10 µg of whole cell extracts (WCE) from NIH 3T3 cells and 32P-labeled site A oligonucleotides (probe A) analyzed under E2F gel shift conditions. Excess unlabeled oligonucleotides (1:150 molar) were used as competitors; A, site A oligonucleotide; E2F, E2F binding site consensus oligonucleotide; C, site C oligonucleotide; mA, mutated site A oligonucleotide. An arrow indicates the probe A–protein complex. (B) Complex formation as above analyzed under alternative gel shift conditions (see Materials and Methods) with 8 µg of WCE. Excess unlabeled oligonucleotides (1:75 molar) were used as competitors; Sp, Sp-1 consensus oligonucleotide; others as in (A). An arrow indicates the probe A–protein complex. (C) Different patterns of mobility shift in sites A and C. The assays were carried out as in (A). An arrow indicates the probe A–protein complex. (D) Complex formation on 32P-labeled site C oligonucleotides. Conditions and competitors are the same as in (A). The upper bracket indicates probe C–protein complexes, representative of E2F bound to site C.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Labeling, Electrophoretic Mobility Shift Assay, Binding Assay, Mobility Shift

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: Promoter activity of two cis-elements enhanced by E2F-1 during the cell cycle. (A) Effect of E2F-1 on the activity of the Dnmt1 core promoter region in NIH 3T3 cells co-transfected with the E2F-1 expression vector pDCE2F-1 and the Dnmt1 promoter constructs. NIH 3T3 cells were co-transfected with either the 300-luc construct (300), the construct with mutated site A (300 A), the construct with mutated site C (300 C) or a construct with mutated sites A and C (300 AC). The ratio of luciferase reporter plasmid, E2F-1 expression vector and pRL-SV internal control vector is 10:10:1. Luciferase activity was determined as described in Figure ​Figure3A.3A. Numbers shown are fold induction relative to luciferase activity of 300-luc in wild-type, parental 3Y1 cells. (B) Activity of intact and mutated Dnmt1 promoters at various stages in the cell cycle. NIH 3T3 cells were transfected with the 300-luc (columns 1, 6, 11 and 16), 300 A-luc (columns 2, 7, 12 and 17), 300 C-luc (columns 3, 8, 13 and 18), 300 AC-luc (columns 4, 9, 14 and 19) and No insert-luc (columns 5, 10, 15 and 20). After 24 h, the cells were arrested by serum starvation, and 36 h later were stimulated by replacement with complete medium. The cells were harvested at the indicated times after serum stimulation for luciferase activity analysis. The luciferase activities are normalized to Renilla luciferase activity. Numbers indicated are luciferase activity relative to the No insert-luc at each time point.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Luciferase

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: HDAC-dependent repression of Dnmt1 transcription. (A) Northern blot showing expression of Dnmt1 mRNA in NIH 3T3 cells 24 h after addition of TSA at the indicated concentrations. Ten micrograms of total RNA were analyzed. (B) Western blot analysis reveals protein levels in NIH 3T3 cells treated with TSA. Twenty micrograms of WCE were subjected to western blot analysis with the indicated antibodies. (C) Cell cycle distribution of NIH 3T3 cells 24 h after addition of TSA at indicated concentrations. DNA content was measured by flow cytometry. (D) Effect of TSA on the activity of the Dnmt1 core promoter region at G0/G1 phase. NIH 3T3 cells were transfected with 300-luc (columns 1 and 6), 300 A-luc (columns 2 and 7), 300 C-luc (columns 3 and 8), 300 AC-luc (columns 4 and 9) or No insert-luc (columns 5 and 10). Twenty-four hours later, cells were growth-arrested by serum starvation for 36 h. Cells were subsequently treated with or without TSA (200 nM) for 24 h before luciferase activity was determined. Luciferase activity is normalized to Renilla luciferase activity and is shown relative to No insert-luc transfections (columns 5 and 10). (E) Effect of TSA on growth-arrested cells. Prior to flow cytometry, NIH 3T3 cells were serum starved for 36 h, and then treated with (+TSA) or without (–TSA) 200 nM TSA for 24 h. Asyn, asynchronized NIH 3T3 cells.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Northern Blot, Expressing, Western Blot, Flow Cytometry, Activity Assay, Transfection, Luciferase

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: The E2F-Rb-HDAC complex decreases the promoter activity of mouse Dnmt1. Saos-2 cells were co-transfected with the plasmids indicated in the figure. The ratio of luciferase reporter plasmid, E2F-1 expression vector, Rb expression vector and pRL-SV40 internal control vector was 10:10:60:1. TSA was added 24 h after transfection. TSA (+) and TSA (++) indicate treatment with concentrations of 100 and 200 nM, respectively. Luciferase activity was determined 48 h post-transfection. The luciferase counts were normalized to Renilla luciferase activity. The standard luciferase activity (100%) was defined as that of cells transfected with the 300-luc and empty expression vectors (column 1).

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

Journal:

Article Title: Transcription of mouse DNA methyltransferase 1 ( Dnmt1 ) is regulated by both E2F-Rb-HDAC-dependent and -independent pathways

doi:

Figure Lengend Snippet: A proposed model for the regulatory mechanisms of mouse Dnmt1 transcription. At all stages of the cell cycle, there is basal level expression of Dnmt1 driven by site A. The E2F site alters transcription by different means, depending on the phase of the cell cycle. At G0/G1 phase Rb can bind to E2F, recruiting HDAC to the complex, resulting in repression of transcription of Dnmt1. A transcription factor bound to site A (TFBA) maintains low transcriptional levels of Dnmt1. At G1/S phase, the inhibition of Dnmt1 transcription mediated by the E2F site is released, and E2F and TFBA now work cooperatively to increase the expression levels in a regulated manner.

Article Snippet: Whole cell extracts were subjected to western blot analysis using anti-Dnmt1 ( 33 ), anti-p21 Waf1 (Santa Cruz) and anti-β-tubulin (Sigma) antibodies.

Techniques: Expressing, Inhibition

Journal: Nature Immunology

Article Title: A SIRT7-dependent acetylation switch regulates early B cell differentiation and lineage commitment through Pax5

doi: 10.1038/s41590-024-01995-7

Figure Lengend Snippet: a , Heatmap displaying the single-cell expression of Sirt1 , Sirt2 , Sirt6 and Sirt7 in the populations (B cells, innate lymphoid cells (ILCs), macrophages (Mac), hematopoietic and progenitor stem cells (HPSC) and T cells) annotated by sc-RNA-seq from purified murine BM Lin − cells. Data was obtained from singlecell.broadinstitute.org (study SCP978). b,c , sc-RNA-seq feature plot ( b ) and single-cell expression of Sirt7 ( c ) in B cells, T cells, NK cells, plasma cells, dendritic cells, plasmacytoid dendritic cells, platelets and erythroid cells of human BM. Data was obtained from singlecell.broadinstitute.org (study SCP101). In panel c , only those cells with detectable counts are plotted, and the dashed lines indicate the median and the first and fourth quartiles. Statistical significance was assessed by one-way ANOVA with Dunnet comparison. d , Microarray expression of Sirt1 (top left), Sirt2 (top right), Sirt6 (bottom left) and Sirt7 (bottom right) in B lymphopoiesis stages (n = 3; common lymphoid progenitors (CLP), n = 2). Data are presented as mean ± s.d. A two-tailed t -test was performed comparing the expression in pooled CLP–pro-B and in pooled pre-B–plasma cells. Data were obtained from the Immgen consortium ( GSE15907 ). e , RNA-Seq expression patterns of Sirt1 , Sirt2 , Sirt6 and Sirt7 in pro-B cells and pre-B cells. Data are presented as the mean of the two replicates (n = 2). f , Gating strategy defining B cell subsets (B220 + CD19 − pre-pro-B cells, B220 + CD19 + IgM − CD43 + pro-B cells, B220 + CD19 + IgM − CD43 − pre-B cells, B220 + CD19 + IgM + immature B cells and B220 hi CD19 + mature B cells) for intracellular flow cytometry measurement of SIRT7 protein levels in fixed mouse BM.

Article Snippet: To understand the role of sirtuins in hematopoiesis, we analyzed the expression levels of sirtuins Sirt1 , Sirt2 , Sirt6 and Sirt7 in Lin − (Ter119 − CD11b − B220 − Gr-1 − ) progenitors using publicly available single-cell RNA-sequencing (scRNA-seq) data from mouse bone marrow (BM) cells (Broad Institute, study SCP978).

Techniques: Expressing, RNA Sequencing, Purification, Clinical Proteomics, Comparison, Microarray, Two Tailed Test, Flow Cytometry

Journal: Nature Immunology

Article Title: A SIRT7-dependent acetylation switch regulates early B cell differentiation and lineage commitment through Pax5

doi: 10.1038/s41590-024-01995-7

Figure Lengend Snippet: a , t -Distributed stochastic neighbor embedding plots displaying the single-cell expression profiles of nuclear sirtuins in purified mouse BM Lin – cells (left) and scRNA-seq feature plot (right) identifying B cells, macrophages, hematopoietic and progenitor stem cells (HPSCs), innate lymphoid cells (ILCs) and T cells. Data were obtained from singlecell.broadinstitute.org (study SCP978). b , Immunoblot of SIRT7 expression in mouse BM CD19 − cells and CD19 + B cells ( n = 3). c , d , Representative histograms ( c ) and quantification of SIRT7 median fluorescence intensity (MFI; d ) in B220 + CD19 – pre-pro-B cells, B220 + CD19 + IgM − CD43 + pro-B cells, B220 + CD19 + IgM − CD43 − pre-B cells, B220 + CD19 + IgM + immature B cells and B220 hi CD19 + mature B cells measured by intracellular flow cytometry ( n = 8 mice). e , Total number of B220 + CD19 + B cells in the BM of wild-type and Sirt7 Δ4–10 129Sv mice ( n = 9). Data were pooled from four independent experiments. f , g , Representative histograms ( f ) and numbers ( g ) of pre-pro-B cell, pro-B cell, pre-B cell, immature B cell and mature B cell populations identified as in c in the BM of 129Sv wild-type and Sirt7 Δ4–10 mice ( n = 9). Data were pooled from four independent experiments. h , i , Representative histograms ( h ) and numbers ( i ) of splenic CD19 + B cells from wild-type ( n = 5) and Sirt7 −/− ( n = 4) mice. j , Hematoxylin and eosin staining of histological sections from the spleens of wild-type and Sirt7 −/− mice ( n = 5); scale bar, 500 µm. k , Number of splenic B220 + CD19 + CD21 + CD23 + CD93 + transitional B cells, B220 + CD19 + CD21 hi CD23 − marginal zone (MZ) B cells, B220 + CD19 + CD21 + CD23 + CD93 − follicular B cells, B220 + CD19 + IgM + Gl7 + Fas + germinal center (GC) B cells, B220 + IgG1 + class-switched B cells and BM CD19 + CD38 + CD138 − Gl7 − memory B cells and B220 lo CD138 + plasma cells from wild-type and Sirt7 −/− mice ( n = 4). l , m , Numbers of donor-derived CD45.1 – CD45.2 + CD19 + B cells in the spleens of recipient CD45.1/CD45.2 mice 4 weeks after congenic transplantation of either CD45.2 wild-type ( n = 4) or Sirt7 −/− ( n = 5) Lin − B220 + CD19 + IgM − pro-B cells expanded ex vivo with OP9 cells and 10 ng ml –1 IL-7, SCF and FLT3-L ( l ) or Sirt7 −/− Lin − B220 + CD19 + IgM − pro-B cells retrovirally expressing empty vector (EV; n = 6), SIRT7 WT ( n = 3) or SIRT7 H187Y ( n = 4; m ). Data are presented as mean ± s.d. ( d , i , and k – m ) or mean ± s.e.m. ( e , g , l and m ) and were analyzed by one-tailed t -test ( e , g , i , k and l ) or one-way analysis of variance (ANOVA) with Sidak multiple comparisons ( d and m ).

Article Snippet: To understand the role of sirtuins in hematopoiesis, we analyzed the expression levels of sirtuins Sirt1 , Sirt2 , Sirt6 and Sirt7 in Lin − (Ter119 − CD11b − B220 − Gr-1 − ) progenitors using publicly available single-cell RNA-sequencing (scRNA-seq) data from mouse bone marrow (BM) cells (Broad Institute, study SCP978).

Techniques: Expressing, Purification, Western Blot, Fluorescence, Flow Cytometry, Staining, Clinical Proteomics, Derivative Assay, Transplantation Assay, Ex Vivo, Plasmid Preparation, One-tailed Test

Journal: EMBO Reports

Article Title: BRD9 is a druggable component of interferon‐stimulated gene expression and antiviral activity

doi: 10.15252/embr.202152823

Figure Lengend Snippet: A Schematic representation of the type I IFN signaling pathway in human cells. B Stimulation of A549/pr(ISRE).eGFP.A1 cells with 1,000 IU/ml of IFN‐α2 for 16 h results in high and homogeneous expression of eGFP as determined by fluorescence microscopy (left panels, scale bars indicate 300 μm) and flow cytometry (right panel). The FITC channel was used to monitor eGFP levels by flow cytometry, and AU refers to arbitrary units. Data are representative of at least two biological replicates. C A549/pr(ISRE).eGFP.A1 cells were transduced for at least 10 days with lentivirus pools expressing Cas9 and sgRNAs targeting the indicated host genes. eGFP levels following 16 h of IFN‐α2 treatment (1,000 IU/ml) or mock were determined by flow cytometry. Data are representative of at least two biological replicates. D Workflow overview of the genome‐scale loss‐of‐function screen for essential IFN signaling components using the full GeCKOv2 CRISPR‐Cas9 library. E 1/αRRA scores from two biological replicates of the genome‐scale screen. Dotted lines represent the significance cutoffs for each replicate. Genes identified as significant in both screens are highlighted. Data information: See also Fig and Dataset .

Article Snippet: Briefly, the GeCKOv2 plasmid library with 6 guide RNAs per human gene was propagated in Endura ElectroCompetent cells (Lucigen), and lentivirus stocks were generated by co‐transfecting 293T cells with the GeCKOv2 plasmid library, pMD2.G and psPAX2 (Addgene plasmids #12259 and #12260, gifts from Didier Trono) at a ratio of 4:2:1 (GeCKOv2:psPAX2:pMD2.G).

Techniques: Expressing, Fluorescence, Microscopy, Flow Cytometry, CRISPR

Journal: EMBO Reports

Article Title: BRD9 is a druggable component of interferon‐stimulated gene expression and antiviral activity

doi: 10.15252/embr.202152823

Figure Lengend Snippet: A A549/pr(ISRE).eGFP.A1 cells were transduced for at least 10 days with lentiviruses expressing Cas9 and individual sgRNAs derived from the GeCKOv2 CRISPR‐Cas9 library targeting BRD9, STAT1, or IFNLR1. eGFP levels following 16 h of IFN‐α2 treatment (1,000 IU/ml), or mock, were determined by flow cytometry. MFI = mean fluorescence intensity. Data represent means and standard deviations from n = 3 biological experiments (individual data points shown). Statistical significance was determined relative to the parental cells stimulated with IFN‐α2 using single‐tailed ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant). B Schematic representations of the known components of the canonical BRG1‐ or BRM‐associated factors (BAF) complex, the Polybromo‐containing BAF (PBAF) complex, and the non‐canonical BAF (ncBAF) complex. Factors reported to be unique to each complex are indicated with colored shapes. BRD9 is unique to the ncBAF complex. C A549‐2D8 cells were transfected for 32 h with the indicated siRNA SMARTpools, or siSCR control, prior to treatment with 0, 10, or 100 IU/ml of IFN‐α2 for 16 h. Cells were then infected with VSV‐GFP at an MOI of 1 PFU/cell and total integrated green fluorescent intensities were collected using the Incucyte live‐cell analysis system over the course of 24 h. Area under the curve (AUC) values for GFP‐Intensity during the 10–24 h period post‐infection were determined. Statistical significance was determined by 1‐way ANOVA on log‐transformed values comparing the siSCR + 100 IU/ml IFN‐α2 condition to each of the other + 100 IU/ml IFN‐α2 conditions (**** P < 0.0001; n.s. not significant). Dotted line is a visual guide for minimum virus replication in siSCR cells in the presence of 100 IU/ml IFN‐α2. Numbers above IFN‐α2‐treated bars indicate their approximate difference to the respective untreated conditions. Data represent means and standard deviations from n = 3 biological experiments (individual data points shown).

Article Snippet: Briefly, the GeCKOv2 plasmid library with 6 guide RNAs per human gene was propagated in Endura ElectroCompetent cells (Lucigen), and lentivirus stocks were generated by co‐transfecting 293T cells with the GeCKOv2 plasmid library, pMD2.G and psPAX2 (Addgene plasmids #12259 and #12260, gifts from Didier Trono) at a ratio of 4:2:1 (GeCKOv2:psPAX2:pMD2.G).

Techniques: Expressing, Derivative Assay, CRISPR, Flow Cytometry, Fluorescence, Transfection, Control, Infection, Cell Analysis, Transformation Assay, Virus