Journal:

Article Title: Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function 1

doi: 10.1104/pp.103.024257

Figure Lengend Snippet: HPLC analysis of tocopherols in wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Tocopherols present in Arabidopsis, maize, and Synechocystis sp. PCC6803 lipid extracts were separated by normal phase HPLC and detected using a fluorescence detector with 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. A, Arabidopsis leaf tissue: solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize leaf tissue: solid line, wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803: solid line, wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1 expressed in the Δslr1737 insertional mutant. Retention times of α-, β-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards. LU, Luminescence units.

Article Snippet: PCR-based markers were designed using INDEL or SNP from the Cereon Arabidopsis Polymorphism and Landsberg erecta Sequence Collection (Cereon Genomics LLC, Cambridge, MA; Jander et al., 2002 ).

Techniques: Mutagenesis, Fluorescence

Journal:

Article Title: Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function 1

doi: 10.1104/pp.103.024257

Figure Lengend Snippet: HPLC analysis of the prenyl quinones from wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Lipids were extracted from Arabidopsis, maize, and Synechocystis sp. PCC6803, and total prenyl quinines were isolated by thin-layer chromatography (TLC) and then analyzed by normal phase HPLC (see “Materials and Methods”) A, Arabidopsis. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize. Solid line, Wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803. Solid line, Wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1cDNA expressed in the Δslr1737 mutant background. Insets, Spectra of the peak labeled DMPBQ. Phyllo, Phylloquinone; PQ, Plastoquinone.

Article Snippet: PCR-based markers were designed using INDEL or SNP from the Cereon Arabidopsis Polymorphism and Landsberg erecta Sequence Collection (Cereon Genomics LLC, Cambridge, MA; Jander et al., 2002 ).

Techniques: Mutagenesis, Isolation, Thin Layer Chromatography, Labeling

Journal:

Article Title: Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function 1

doi: 10.1104/pp.103.024257

Figure Lengend Snippet: HPLC analysis of seed tocopherols in wild-type Arabidopsis, vte1-1, and vte1-2. Total seed lipids were extracted, and the tocopherols present were separated by reverse phase HPLC and detected using a fluorescence detector; 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. Retention times of α-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards.

Article Snippet: PCR-based markers were designed using INDEL or SNP from the Cereon Arabidopsis Polymorphism and Landsberg erecta Sequence Collection (Cereon Genomics LLC, Cambridge, MA; Jander et al., 2002 ).

Techniques: Fluorescence

Journal:

Article Title: Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function 1

doi: 10.1104/pp.103.024257

Figure Lengend Snippet: TC activity of proteins expressed in E. coli. E. coli cell lysates from cells overexpressing the empty pET vector or pET engineered to express TC proteins from Arabidopsis, maize, and Synechocystis sp. PCC6803 were incubated with radiolabeled 2,3-methyl-6-phytyl-1,4-benzonequinol (3 methyl 14C) for 4 h as described in “Materials and Methods.” Total lipids were extracted, separated by TLC, and radiolabeled products were detected by phosphor imager analysis. Products were identified by comigration with standards. The 14C incorporation into γ-tocopherol was quantified densitometrically and expressed as pixels per microgram of total protein.

Article Snippet: PCR-based markers were designed using INDEL or SNP from the Cereon Arabidopsis Polymorphism and Landsberg erecta Sequence Collection (Cereon Genomics LLC, Cambridge, MA; Jander et al., 2002 ).

Techniques: Activity Assay, Plasmid Preparation, Incubation

Journal:

Article Title: Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function 1

doi: 10.1104/pp.103.024257

Figure Lengend Snippet: Analysis of Glc, Suc, and starch in wild-type Arabidopsis and vte1-1 Glc, Suc, and starch levels were analyzed spectrophotometrically using the enzyme-coupled assays described in “Materials and Methods.” Glc and Suc are expressed as nanomoles per milligram fresh wt ( n = 4).

Article Snippet: PCR-based markers were designed using INDEL or SNP from the Cereon Arabidopsis Polymorphism and Landsberg erecta Sequence Collection (Cereon Genomics LLC, Cambridge, MA; Jander et al., 2002 ).

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 1. Rbm24 regulates differentiation of ESCs. (A): Expression profiles of pluripotency markers (Oct4 and Nanog), mesodermal markers (Mesp1 and T-Bra), Rbm24, and cardiac marker (Nkx2.5) during normal ESC differentiation by embryoid body (EB) formation as measured by quantitative real-time polymerase chain reaction. Data were normalized to Gapdh. Error bars represent mean 6 SD from three biologically independent experiments. (B): Schematic diagram depicting the methodology for generating an ESC line for inducible expression of Rbm24 by DOX. Upper panel: Inducible cassette exchange recombination in A2Lox ESCs (Supporting Information Fig. S1A); lower panel: experimental procedure. (C): Rbm24-IRES-GFP expression following induction of A2lox ESCs with doxycycline (1Dox) for 48 hours. No GFP expression was detected in uninduced cells (2Dox). Scale bar5 200 lm (Supporting Information Fig. S1B for 24-hour Dox induction). (D): Kinetics of Rbm24 expression using Western blot with anti-Rbm24 antibody. Rbm24 was almost undetectable in uninduced condition but was rapidly upregulated following Dox stimulation. (E): GFP expression as measured by flow cytometry after Dox induction for 48 hours in ESCs. (F): Overexpression of Rbm24 for 3 days by Dox induction leads to an altered morphology of ESCs. (G): Alkaline phos- phatase staining and (H) immunofluorescence staining of Oct4 at day 3 following induction of Rbm24 by Dox in the absence of feeder cells and LIF. Scale bar5 200 lm. Abbreviations: Dox, doxycycline; ESC, embryonic stem cell; GFP, green fluorescent protein; T-Bra, T-brachyury.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Cytometry, Over Expression, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 2. Rbm24 overexpression promotes cardiac differentiation of ESCs. (A): Schematic overview of experimental strategy. Assays were performed at different time points as described in the Results section are as indicated in the diagram. (B): Quantification of pluri- potency markers (Oct4, Nanog, and Lefty1), lineage markers (Nestin, Sox1, Sox17, AFP, T-bra, and Mesp1), and cardiac markers (Nkx2.5, Mef2c and Gata4) analyzed by qRT-PCR after 48 hours of Rbm24 induction in ESC medium in the absence of leukemia inhibitory factor. Note that Rbm24 induces the expression of cardiac mesoderm markers Mesp1 and T-bra significantly. Data represent relative expression of transcripts compared to untreated ESCs as mean 6 SD of three biologically independent experiments. (*, p < .05; **, p < .01; ***, p < .001). (C): Rbm24 overexpression promotes cardiac commitment in EB cultures as detected by the precocious appearance and enhancement of beating areas, compared to uninduced cells (Supporting Information video). Error bars represent mean 6 SD from five biologically independent experiments. (D): Immunofluorescence staining of beating cells. Day 11 EBs were dissociated and plated on chamber slides and stained with Nkx2.5, Tpm, Actn2, Tnnt2, Myh6, Mlc2a, and Mlc2v antibodies, the specific markers for cardiomyocyte. Scale bar 5 200 lm. (E): Quantification of cardiomyocytes by flow cytometry. At day 12 after cardiac differentiation of ESCs, EBs were dissociated and stained for cardiac-specific marker Actn2. (F): Patch-clamp electrophysiology showed representative ventricular-like, atrial-like, and nodal-like action potentials of ESC-derived cardiomyocytes. Beating EBs were enzymatically dissociated to single cells and action potentials (APs) were assessed. Types of APs were recorded in cardiomyocytes isolated from 19 days EBs after differentiation with Rbm24 induction. The same types of APs were recorded in control cells. Abbreviations: Dox, doxycycline; EB, embryoid body; ESCs, embryonic stem cells; FACS, fluorescence activated cell sorting; qPCR, quantitative polymerase chain reaction.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Staining, Cytometry, Marker, Patch Clamp, Derivative Assay, Isolation, Control, FACS, Real-time Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 3. RNA-seq analysis of gene expression regulated by Rbm24. (A): A scatter plot depicting the differentially expressed genes fol- lowing Dox induction. Red dots represent upregulated genes, and green dots represent downregulated genes. (B): Gene ontology analy- sis of upregulated genes after 48 hours Rbm24 induction. (C): Gene ontology analysis of downregulated genes after 48 hours Rbm24 induction. Abbreviation: Dox, doxycycline.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: RNA Sequencing, Gene Expression

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 4. RNA-seq analysis of alternative splicing (AS) events regulated by Rbm24. (A): Overview of algorithm to analyze Rbm24- regulated AS events. Sequences of cDNA fragments generated from high throughput sequencing were aligned to the genome by a spliced aligner (Tophat) [22]. Cufflinks was then used to assemble transcript structures and estimate transcript abundance [23]. (B): Validation of RNA-seq data by real-time polymerase chain reaction (RT-PCR). Naca has two isoforms, skNac and aNac, generated by alternative splicing a 6 kb exon 2. Two primer pairs were used to amplify these two isoforms, respectively. Hprt was used as an internal control. 30 cycles of PCR were performed. (C): Gene ontology analysis of Rbm24-regulated AS events (molecular function). (D): Gene ontology analysis of Rbm24-regulated AS events (biological process). (E): Gene ontology analysis of Rbm24-regulated AS events (cellular component). Abbreviation: Dox, doxycycline; FPKM, fragments per kilobase of transcript per million fragments mapped.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: RNA Sequencing, Alternative Splicing, Generated, Next-Generation Sequencing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 5. Rbm24-regulated alternative splicing (AS) events are related to cardiac differentiation. (A): Rbm24-regulated AS events are enriched in the mitochondrion and cytoskeleton. (B): Real-time polymerase chain reaction (RT-PCR) analysis of Rbm24-regulated AS events in ESCs with or without Rbm24 induction. RNA was isolated 48 hours after Dox induction. Hprt was used as an internal control. Schematics of all splicing events are shown. i1-exclusion isoform (exon excluded); i2-inclusion isoform (exon included). Upregulation of i2 isoform of Tpm1, Capzb, Tpm3, Itga6, Sun1, and skNac was detected upon Dox treatment. Atp5c1 displayed exon exclusion AS pat- tern, i1 was upregulated by Rbm24. (C): Time-course analysis of AS events by RT-PCR during A2Lox ESC differentiation toward cardiomy- ocytes. Differential expression of AS events (observed in Fig. 5B) was detected in EBs 6 and 12 days after cardiac differentiation. i1/i2 are annotated as per Figure 5B. Hprt was used as an internal control. Abbreviations: DOX, doxycycline; ESC, embryonic stem cell.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: Alternative Splicing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Quantitative Proteomics

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 6. Rbm24 is required for embryonic stem cell (ESC) cardiogenesis. (A): Quantitative real-time polymerase chain reaction (qRT- PCR) of day 6 and 12 EBs after ESC (R1 line) cardiac differentiation showed efficiency of Rbm24 shRNA-lentivirus knockdown (KD) at transcript level. Data were normalized to Gapdh. Error bars represent mean 6 SD from three biologically independent experiments (***, p < .001). (B): Western blot of day 0, 3, 6, 9, and 12 EBs after cardiac differentiation showed depletion of Rbm24 protein in shRNA KD cells. Gapdh was used as an internal control. (C): Rbm24 KD inhibited ESC cardiac differentiation as demonstrated by barely detectable beating EBs in shRNA KD cells compared to control. Error bars represent mean 6 SD from three biologically independent experiments. (D): Temporal analysis of marker expression in ESC differentiating over 12 days in Rbm24 KD versus control cells by qRT-PCR. Data were normalized to Gapdh. Error bars represent mean 6 SD from three biologically independent experiments. (E): AS patterns of day 0, 3, 6, and 12 EBs after cardiac differentiation were compared between Rbm24 KD cells and control cells by RT-PCR. i1/i2 are annotated as per Figure 5B. Gapdh was used as an internal control. Abbreviation: EB, embryoid body.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, shRNA, Knockdown, Western Blot, Control, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

doi: 10.1002/stem.2366

Figure Lengend Snippet: Figure 7. Rbm24 regulates alternative splicing of pluripotency genes. (A): Venn diagram showing the overlap of hESC identity mainte- nance genes with genes splicing regulated by Rbm24. Genes essential for hESC maintenance were extracted from Supporting Informa- tion Table S1 of genome-wide RNAi screen in hESCs [34]. (B): Diagram of Tpm1 splicing patterns in ESCs versus tissues, primers designed for qRT-PCR detection are indicated by black arrows. (C): Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicates a Tpm1 splicing isoform switch during cardiac differentiation of ESCs. Data were normalized to Gapdh. Note the alternation of expression levels between two isoforms during ESC differentiation as indicated by the decreased ratio of mRNA with exon5 versus exon6. (D): Rbm24 decreased the expression of ESC-specific exon 5 and increased the expression of tissue-specific exon 6. qRT-PCR was used to measure the mRNA level. Data were normalized to Gapdh (n 5 3; **, p < .01). (E): qRT-PCR was used to determine efficiency and specificity of isoform-specific knockdown in ESCs. Data were normalized to Gapdh. The expression levels of Tpm1-E5 or Tpm1-E6 were significantly decreased by E5 or E6 siRNA, respectively (n 5 3, **, p < .01; ***, p < .001). (F): The effect of Tpm1 isoform- specific knockdown in ESCs as determined by alkaline phosphatase staining (up panel) and immunofluorescence staining of Oct4 (lower panel), observed at day 3 after electroporation of isoform-specific siRNA. Nuclei were stained with Hoechst (blue). Scale bar 5 200 lm. Abbrevi- ations: Dox, doxycycline; hESC, human embryonic stem cell.

Article Snippet: R1 ESCs (ATCC, Manassas, VA, http://www.atcc.org) were infected with lentivirus expressing short hairpin RNA (shRNA) targeting mouse Rbm24 gene (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com) and selected with puromycin for 4 days, nontargeting viral particles were used as a control (Ctl).

Techniques: Alternative Splicing, Genome Wide, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Staining, Electroporation

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Heatmap of log 2 FC values for ubiquitylated peptides in MDS-L FBXO11-KO versus WT cells. Shown are hits with a FC of greater than |0.5|. Known RNA-binding proteins are labeled in red. Data were reanalyzed from ref. . ( B ) Scatter plot of the results from co-IP and MS identification of endogenous FBXO11 complexes in F-36P cell nuclear fractions. Shown are the –log( P value) against the FC enrichment of individual proteins identified in the FBXO11 IP versus the IgG control IP. The blue box highlights proteins with a P value of less than 0.05 and a FC of greater than 1.5. The P value was derived by G test. ( C ) STRING network analysis of FBXO11-interacting proteins identified by MS. Only input nodes are shown, with the threshold cutoff of medium confidence at 0.7. The thickness of the lines connecting nodes indicates the relative strength of evidence supporting the protein-protein interactions. ( D ) Strategy for the FBXO11 substrate-focused CRISPR/Cas9 screen, in which the gRNA library encompasses all of the differentially ubiquitylated peptides identified from the ubiquitin proteomics experiment represented in A . ( E ) Summary table of significant hits from the MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout) analysis of the CRISPR screen in D , demonstrating selective enrichment or depletion of guides in sgCTRL or sgFBXO11 colonies. ( F ) Schematic overview of the integrated multiomics approach to identify relevant candidate FBXO11 substrates meeting the listed criteria, revealing NPM1 and HNRNPU. ( G ) FC in individual gRNA read counts for NPM1 and HNRNPU in the colony-forming assay (day 10) versus initial representation (day 0) in the CRISPR screen. The FC for individual guides was compared between sgCTRL and sgFBXO11 experimental groups, shown on the graphs. n = 6 guides per gene, in 2 independent biological replicates of the CRISPR screen. * P < 0.05, by 2-tailed, paired t test.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: RNA Binding Assay, Labeling, Co-Immunoprecipitation Assay, Control, Derivative Assay, Protein-Protein interactions, CRISPR, Ubiquitin Proteomics, Genome Wide, Knock-Out

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of overexpressed FLAG-FBXO11 isoforms in HEK293T cells. Blotting was done for endogenous NPM1 and H2A. Endogenous NPM1 IPs were probed for FBXO11 and NPM1. ( B ) Immunoblots of co-IP FLAG-FBXO11 complexes of FBXO11 and NPM1. ( C ) Left: Input immunoblots for FLAG-FBXO11, NPM1, ubiquitin-GFP, and H2A in HEK293T cells. IPs of NPM1 were performed to detect ubiquitylation by FBXO11. Right: NPM1 versus IgG control IP, immunoblotted for GFP-ubiquitin. n = 3. ( D ) Densitometry for NPM1 poly-ubiquitin bands from C . The area quantified is 75 kDa and above. n = 3. * Q < 0.05, by Kruskal-Wallis ANOVA corrected for multiple comparisons by the Benjamini method. ( E ) Schematic depicting K248-Ub of NPM1 in its C-terminal core. The ubiquitin (Ub) footprint was identified by MS, and . ( F ) HEK239T cells were transfected with NPM1-GFP or NPM1-K248R-GFP fusions. Red outlines indicate GFP-bright regions annotated in QuPath. Scale bars: 10 μm. ( G and H ) Annotated regions quantified for circularity ( G ) and aspect ratio ( H ). n = 2. Dots represent individual NPM1 + regions.* P < 0.05 and ** P < 0.01, by 2-tailed Mann-Whitney U test. ( I ) Confocal images of human CD34 + cells expressing sh CTRL or sh FBXO11 mCherry constructs. Images are pseudocolored for FBXO11 (magenta), NPM1 (green), and DAPI (blue). Scale bars: 5 μm. ( J ) NPM1-bright objects per cell. * P < 0.05, by 2-tailed Mann-Whitney U test. ( K ) Circularity values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( L ) Aspect ratio values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( M ) Relative MFI values for FBXO11 signal and NPM1 signal on a per-cell basis. **** P < 0.0001, by 2-tailed t test. ( N ) Human CD34 + cells stained for FBXO11 (green) and NPM1 (magenta). Colocalization (white arrowheads) where green and magenta overlap. Scale bars: 5 μm. ( O ) Pearson correlation values for NPM1 and FBXO11 signals in the nucleolus (NPM1-bright) and nucleoplasm (NPM1-dim). Each dot represents 1 cell, with greater than 100 cells at ×63. **** P < 0.0001, by 2-tailed Mann-Whitney U test.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Control, Transfection, MANN-WHITNEY, Expressing, Construct, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Schematic of splicing reporter delivered to F-36P cells. ( B ) Immunoblot for FBXO11 in HEK293T lysates with an increasing transfected plasmid dose of FBXO11-long and FBXO11-short, compared with endogenous hnRNPR and SYNCRIP. ( C ) Percentage of DsRed + cells normalized to reporter-positive cells in B measured by FACS at 48 hours. * Q < 0.05 and ** Q < 0.01, by multiple 2-tailed t tests corrected for multiple comparisons. ( D ) Representative flow plots of FBXO11-long and FBXO11-short reporter-positive cells. ( E ) DsRed-eGFP + F-36P cell percentages normalized to reporter-positive cells. n = 3 independent experiments; n = 3 wells per group. ** P < 0.01, by 2-tailed t test. ( F ) FC in DsRed + cells in sgFBXO11 versus sgNT, in the context of parental F-36P cells or F-36P cells with NPM1 overexpression. ** Q < 0.01. ( G ) Representative flow plots of sgFBXO1 versus sgNT in F-36P cells overexpressing NPM1. ( H ) log 2 FC of FBXO11 expression in healthy control cells or MDS CD34 + samples, grouped by common splicing factor mutations. SF WT, splicing factor WT. * Q < 0.05, by 1-way ordinary ANOVA corrected for multiple comparisons using the Benjamini method. ( I ) Number of significant alternative splicing events determined by rMATS analysis. ( J ) Top: Schematic of junction reads versus skipped reads, used to determine exon inclusion levels for splicing events. Bottom: Exon inclusion levels for putative FBXO11-dependent splicing events in SLC22A16 and AFTPH in MDS CD34 + samples that were null for any splicing factor mutation. Each dot represents 1 patient.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Western Blot, Transfection, Plasmid Preparation, Over Expression, Expressing, Control, Alternative Splicing, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Workflow to isolate CD34 + cell fractions from healthy donor ( n = 6) and MDS patient ( n = 13) samples, to normalize to 25,000 live cells per replicate tube, and perform total quantitative proteomics by DIA of peptide spectra. Illustration was created in BioRender.com. ( B ) Volcano plot of differentially detected proteins in MDS samples versus healthy controls. Each data point indicates 1 protein, with coordinates reflecting the –log 10 of the P value against its log 2 FC for patients with MDS ( n = 13) versus healthy donors ( n = 6). Color-coding indicates a P value cutoff of less than 0.05 and a log 2 FC cutoff >|0.5|. ( C ) Heatmap of the top 400 differentially expressed proteins in MDS versus healthy controls. Unsupervised hierarchical clustering of patient samples was performed using ClustVis. Each column represents 1 replicate tube from the indicated patient samples below the map. R1, run 1; R2, run 2. ( D ) GO molecular function analysis of cluster 2 proteins, the largest cluster downregulated in MDS. ( E ) GO molecular function analysis of cluster 8 proteins, the largest cluster upregulated in MDS. ( F ) Schematic depicting the data integration performed using Cytoscape to quantify the FBXO11 interactome in primary MDS HSPCs. Schematic was created in BioRender.com. ( G ) Resultant data visualization of the integrated proteomics analysis performed in F . Blue indicates down in MDS; red indicates up in MDS. ( H ) Normalized enrichment scores from GSEA of the significantly differentially expressed proteins in the MDS proteome versus healthy controls.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Quantitative Proteomics

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Upstream regulator analysis of FBXO11-interacting proteins performed using IPA software. MYC was identified as a significant upstream regulator of the FBXO11 interactome ( P = 1.07 × 10 15 ). Shown is the MYC mechanistic network in hierarchical format, with FBXO11-interacting proteins in blue. ( B ) Integrative genomics viewer gene track of FBXO11 from MYC ChIP-Seq results in ENCODE. ( C and D ) FC of MYC binding to FBXO11 promoter sequences, calculated from MYC ChIP-qPCR in F-36P and MDS92 cells. TLR2 agonist (CU-T12-9) treatment resulted in a complete loss of MYC expression. * Q < 0.05, by 2-way ANOVA corrected for multiple comparisons using the Benjamini method. ( E ) TLR2 peptide intensity determined by quantitative proteomics from MDS patient sample replicates in which TLR2 peptides were detected. Each dot indicates 1 replicate. **** P < 0.0001, by 2-tailed Mann Whitney U test.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Software, ChIP-sequencing, Binding Assay, ChIP-qPCR, Expressing, Quantitative Proteomics, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of FBXO11, NPM1, and H3 with densitometry. Each sample was normalized to H3, and then to controls. ( B ) Densitometric values of FBXO11 and NPM1 from A . Pearson correlation for all pairs of values and line of best fit with a 95% CI. ( C ) Cell fitness assay using primary CD34 + cells. FC in indel percentages at the end of the assay versus the initial read. For double knockout, the indel percentage tracks NPM1 edits. * Q < 0.05 and ** Q < 0.01, by 2-group specific longitudinal mixed-effects analysis corrected for multiple comparisons. ( D ) Number of colonies. * Q < 0.05, ** Q < 0.01, and *** Q < 0.001, by multiple t tests corrected for multiple comparisons. n = 3. ( E ) Number of colonies. n = 62 with 3 wells per assay. * Q < 0.05 and ** Q < 0.01, by t tests on pooled replicates corrected for multiple comparisons. ( F ) Growth curve of F-36P GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( G ) Growth curve of MDS92 GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( H ) Schematic of the RUNX1-driven MDS mouse model on an inducible Mx1-Cre + Fbxo11 +/+ or Fbxo11 +/– background. ( I ) Percentage of GFP + peripheral blood cells isolated from Fbxo11 +/+ RUNX1-GFP or Fbxo11 +/– RUNX1-GFP transplants. n = 7–10 mice per group. **** P < 0.0001, by fixed-effects (type III) analysis. ( J ) Percentage of GFP + mononuclear cells isolated from BM aspirates of transplant recipients at 11 weeks. n = 8–9 mice per group. * P -linear < 0.05, by 2-tailed t test. ( K ) Percentage contribution to the LSK, Lin - /Sca1 + /Kit + /SLAM + (signaling lymphocyte activation molecules) (LSK-SLAM + ) gate of immunophenotypically defined HSPCs in surviving RUNX1 transplant recipients. n = 6 mice per group. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP-GM, granulocyte-monocyte biased multipotent progenitors; MPP-MkE, megakaryocyte-erythroid biased multipotent progenitors; MPP Ly, lymphoid-biased multipotent progenitor. Q > 0.05, by t tests corrected for multiple comparisons (no significant differences were detected). ( L ) Percentage contribution of RUNX1-GFP + common myeloid progenitor (CMP), GMP, and megakaryocyte-erythrocyte progenitor (MEPs) in the Lin – c-kit + HSPC compartments. n = 6 mice per group. * P < 0.05, by multiple unpaired tests, t test for groups with normal distribution, or Mann-Whitney U test. ( M ) Differential CBCs in mice 16 weeks after pIpC. * P < 0.05, by unpaired, 2-tailed t for groups with normal distribution or Mann-Whitney U test. ( N ) Strategy of Nup98-Hoxd13 -driven MDS mouse model with sh CTRL or sh Fbxo11 vectors. n = 6–10 mice per group. ( O ) Western blot for FBXO11, NPM1, and ACTIN in c-kit + cells from Nup98-Hoxd13 + mice, transduced with lentiviral sh CTRL or sh Fbxo11 . ( P ) CBC in Nup98-Hoxd13 transplants at 2 months. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.005, by unpaired, 2-tailed t test for groups with normal distribution or Mann-Whitney U test. ( Q ) BM cellularity of Nup98-Hoxd13 mice. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( R ) Representative H&E-stained femur cells from Q . Original magnification, ×10. Scale bar: 200 μm.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Western Blot, Double Knockout, Plasmid Preparation, Isolation, Activation Assay, MANN-WHITNEY, Transduction, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Map of FBXO11 mutations identified by whole-exome sequencing of the MSK-IMPACT cohort of patients with hematologic malignancies. ( B ) Alpha-fold predicted structure of FBXO11 with structural features and mutations within myeloid diseases mapped in the disordered N-terminus. ( C ) Variant allele frequencies plotted along the amino acid residues affected by FBXO11 mutations; FBXO11 functional domains are boxed. N-term., N-terminus; ZnF-UBR, zinc finger-ubiquitin-protein ligase E3 component n-Recognin 1. ( D ) Prediction of intrinsically unstructured proteins 2 (IUPRED2) score of amino acid residues in FBXO11-long. The blue arrowhead indicates the initial methionine residue in FBXO11-short. ( E ) Representative confocal images of FBXO11-long and FBXO11-short expressed in HEK293T cells. Scale bars: 10 μm. ( F ) Quantification of signal distribution of FBXO11-long + and FBXO11-short + cells using the SD of FLAG-FBXO11 signal across each nucleus. ** P < 0.01, by 2-tailed, unpaired Mann-Whitney U test. ( G ) GSEA analysis of RNA-Seq data ( GSE156708 ) from MDS-L cells that were FBXO11-KO compared with MDS-L parental cells, cells with reexpression of FBXO11-long cDNA, or cells with reexpression of FBXO11-short cDNA. The size of circle indicates the number of genes in the set.

Article Snippet: Mouse shRNA constructs targeting Fbxo11 were purchased from Origene (pLenti-GFP vector).

Techniques: Sequencing, Variant Assay, Functional Assay, Ubiquitin Proteomics, Residue, MANN-WHITNEY, RNA Sequencing

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: Illustrating three genome-wide significant loci conferring risk of sCJD (in PRNP, STX6 and GAL3ST1 ) and two genes significant only with gene-wide tests ( PDIA4, BMERB1 ).

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Genome Wide

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: CAVIAR utilizes summary statistics and LD structure to predict the probability of each variant being causal, producing a ‘causal set’ with 95% probability of containing the causal SNP, whilst allowing for the possibility of multiple causal SNPs at each locus. Each locus was defined as 100 variants upstream and downstream of the top SNP. Plots show causal posterior probability of each variant at ( a ) PRNP , ( b ) STX6 and ( c ) GAL3ST1 coloured by LD (1000G; EUR) with the top SNP. Circles indicate variants within the 95% causal set.

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Variant Assay

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: Plot of –log 10 of P-values from the GWAS association analysis at the STX6 locus (black) and the eQTL association analysis from the GTEx dataset (v7) (red) for: ( a ) STX6 expression in the caudate, ( b ) STX6 expression in the putamen, ( c ) STX6 expression in the hypothalamus, ( d ) KIAA1614 expression in the tibial nerve, ( e ) KIAA1614 expression in the tibial artery. Peaks correspond to the CLPP in the eCAVIAR analysis with a higher degree of colocalisation with increasing CLPP (see Supplementary Table 4).

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Expressing

Journal: medRxiv

Article Title: Genome-wide association study identifies risk variants for sporadic Creutzfeldt-Jakob disease in STX6 and GAL3ST1

doi: 10.1101/2020.04.06.20055376

Figure Lengend Snippet: N2aPK1/2 cells were transfected with pRetroSuper vectors containing Stx6 (‘shSTX6 1’, ‘shSTX6 2’) or Prnp (‘shPRNP’) targeting shRNAs to generate knockdown cell lines or a scrambled non-silencing (‘shScramble 1’, ‘shScramble 2’) shRNA sequence for controls. Samples were taken prior to plating for SCA for immunoblot (n = 3) and expression normalised to untransfected N2aPK1/2 (indicated by dashed line). ( a-b ) Knockdown of syntaxin-6 protein levels determined by ( a ) immunoblot with anti-syntaxin-6 antibody with ( b ) band intensity measured relative to β-actin loading control (Student’s t-test). ( c-d ) Knockdown of PrP C protein determined by ( c ) immunoblot with anti-PrP antibody ICSM18 with (d) band intensity measured relative to β-actin loading control (Student’s t-test). (e) Average spot count of infected cell number post-4 th split in SCA following infection with RML at 3 × 10 −6 dilution (one-way ANOVA with Tukey’s post-hoc test on log-transformed data). Statistical associations of knockdown lines relative to controls indicated; other results omitted for clarity. All error bars show mean ± SEM; *P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: 3.5 μg of pRetroSuper plasmid containing shRNA targeting mouse Stx6 (5’-TGGAATGCTGGAGTGGCAGATCGCTATGG; Origene), Prnp (5’-GAGACAATCTAAACATTCT; as described previously ) or a non-effective scrambled 29mer (5’-GCACTACCAGAGCTAACTCAGATAGTACT; Origene).

Techniques: Transfection, Knockdown, shRNA, Sequencing, Western Blot, Expressing, Control, Infection, Transformation Assay

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Protein domain structure of EEN as well as a multiple amino acid sequence alignment of EEN, IES6 and human IES6 (hIES6). The YL1-C domain is indicated in blue. ( B ) Aggregated H3K27me3 profile of 2369 Group I genes shows H3K27me3 occupancy from 1 kb upstream to 2 kb downstream of the TSS in L er , ein6-1 , een-1 and ein6-1 een-1 seedlings. ( C ) Genome browser screenshot shows differential enrichment of H3K27me3 at an example Group I gene and Group II gene. To ensure an accurate comparison of individual chromatin features between genotypes, the tracks were normalized to the respective sequencing depth. ( D ) H3K27me3 occupancy of 54 Group II genes in L er , ein6-1 , een-1 and ein6-1 een-1 seedlings is shown as an aggregated H3K27me3 profile from 1 kb upstream to 2 kb downstream of the TSS. The H3K27me3 occupancy was calculated as the ratio between the two respective merged ChIP replicates and the two merged L er IgG control replicates. ( E ) Quantification of H3K27me3 levels in the 5’UTR intron, gene body and 3’UTR of the EIN2 gene are shown. The H3K27me3 occupancy in these regions was calculated as the ratio between the respective merged ChIP-seq samples and merged L er IgG control samples. ( F ) Spearman’s correlation plot shows correlation of read coverages between the antibody validation H2A.Z datasets from this study (Col-0, pie1-1 , Col-0 HTA11:HTA11-GFP (αH2A.Z) and Col-0 HTA11:HTA11-GFP (αGFP)) and three publicly available H2A.Z ChIP-seq datasets ( ; ; ). Clustering is determined by the degree of correlation. ( G ) Heatmap shows the H2A.Z occupancy at all Arabidopsis genes in the indicated genotypes. Levels of H2A.Z from 1 kb upstream to 2 kb downstream of the TSS are shown. ( H ) Genome browser screenshot shows differential enrichment of H2A.Z in Col-0, pie1-1 and Col-0 HTA11:HTA11-GFP seedlings. Moreover, H2A.Z enrichment is also shown for three publicly available H2A.Z ChIP-seq datasets ( ; ; ). Genetic background and used antibodies are indicated. The Col-0 IgG track serves as a control and the L er 5mC track indicates methylated cytosines (CG in yellow, CHG in blue, CGG in pink). The shape difference of H2A.Z domains in the dataset can be explained by the MNase treatment of the chromatin. ( I ) Levels of H2A.Z in the 5’UTR intron, gene body and 3’UTR of EIN2 in L er , ein6-1 , een-1 and ein6-1 een-1 seedlings are shown. The H2A.Z occupancy in these regions was calculated as the ratio between the respective merged ChIP-seq samples and merged L er IgG control samples.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: Sequencing, Comparison, Control, ChIP-sequencing, Biomarker Discovery, Methylation

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Genome browser screenshot visualizes the levels of the depicted chromatin features at the EIN2 gene in untreated 3-day-old etiolated L er , ein6-1 , een-1 and ein6-1 een-1 seedlings. Occupancy of H3K27me3, H2A.Z, H3K4me3 and H2Aub was determined with ChIP-seq, mRNA expression was measured with RNA-seq and levels of methylated cytosines (CG in yellow, CHG in blue, CGG in pink) were determined with MethylC-seq. To ensure an accurate comparison of individual chromatin features between genotypes, the tracks were normalized to the respective sequencing depth. Normalization was separately done for each chromatin feature. Biological replicate 1 of the H3K27me3 and H2A.Z ChIP-seq datasets is shown. ( B ) Venn diagram illustrates the overlap between genes that show a significant increase of H3K27me3 (2-fold enrichment over ein6-1 ), H2AZ (2-fold enrichment over een-1 ) and H2Aub (2-fold enrichment over L er ) in ein6-1 een-1 mutants and also a significant decrease of H3K4me3 (1.5-fold enrichment in L er over ein6-1 een-1 ). In addition, genes that contain differentially methylated regions (DMRs) with ten or more methylated cytosines in at least one genotype were included as well. ( C ) Graphical illustration of H3K27me3 and EIN6 occupancy at the EIN2 gene determined with ChIP-seq. Sequencing reads were merged between biological replicates for the H3K27me3 ChIP-seq using untreated 3-day-old etiolated L er (gray) and ein6-1 een-1 (blue) seedlings (two replicates each) and the EIN6 ChIP-seq (red) using L er 35S:EIN6-FLAG seedlings (three replicates). The occupancy was calculated as the ratio between the respective merged ChIP and the merged L er IgG control in 100 bp bins from 2.4 kb upstream to 7.7 kb downstream of the transcriptional start site (TSS) of EIN2 and is shown as log 2 fold change. Negative values which reflect lower occupancy in the ChIP sample compared to the IgG control sample were set to zero.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Methylation, Comparison, Sequencing, Control

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Histochemical localization of GUS expression driven by a 2 kb EEN promoter fragment in 7-day-old L er EEN:GUS seedlings. ( B ) GFP fluorescence visualizes subcellular EEN localization in roots of 10-day-old Col-0 35S:GFP-EEN seedlings using confocal laser scanning microscopy. 20 µm scale bar is shown. ( C ) Genome browser screenshot visualizes the in vivo binding of EEN to the gene body of EIN2 , At4g11280 and At4g01250 . H2A.Z occupancy in Col-0 and ref6-1 een-2 seedlings at these loci is indicated as well. ( D ) Mass spectrometry analysis of immuno-purified FLAG-tagged EEN from Col-0 35S:EEN flower tissue revealed ARP5 as an interactor. Results from two independent IPs are shown. Flower tissue of non-transgenic Col-0 was used as a control. Both IPs also detected the helicase RVB2 which is part of the yeast INO80 complex as an interaction partner of EEN. ( E ), ( F ) Gene models of EEN and INO80 indicate the localization of the T-DNA insertion in een-2 ( E ), ino80-1 ( F ) and ino80-8 ( F ) mutants. ( G ), ( H ) Expression of EEN ( G ) and INO80 ( H ) in untreated 3-day-old etiolated Col-0, een-2 and ino80-8 seedlings using RNA-seq is shown. Expression is measured in TPM (Transcripts Per Kilobase Million) and results from two biological replicates are shown (mean ±S.D.). ( I ) ( J ) Quantification of H3K27me3 levels ( I ) and H2A.Z levels ( J ) in the 5’UTR intron, gene body and 3’UTR of the EIN2 gene in untreated 3-day-old etiolated Col-0, ref6-1 , een-2, ref6-1 een-2, ino80-1, ref6-1 ino80-1, arp5-1 and ref6-1 arp5-1 seedlings. Occupancy of H3K27me3 and H2A.Z was determined by ChIP-seq. The H3K27me3 and H2A.Z occupancy in the three regions was calculated as the ratio between the respective ChIP-seq sample and Col-0 IgG control sample. ( K ) Triple response phenotype of 3-day-old etiolated Col-0, ref6-1 , een-2, ref6-1een-2, ino80-1, ref6-1 ino80-1 , arp5-1 , ref6-1 arp5-1 , ein2-5 , pie1-1 and ref6-1 een-2 pie1-1 seedlings. Seedlings were grown on control LS media or on LS media supplemented with 10 µM ACC.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: Expressing, Fluorescence, Confocal Laser Scanning Microscopy, In Vivo, Binding Assay, Mass Spectrometry, Purification, Transgenic Assay, Control, RNA Sequencing, ChIP-sequencing

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Graphical representation of H2A.Z and EEN occupancy at the EIN2 gene determined with ChIP-seq. Sequencing reads of two merged H2A.Z ChIP-seq’s (L er (gray), ein6-1 een-1 (blue)) and one EEN ChIP-seq (Col-0 35S:EEN-FLAG (red)) using 3-day-old etiolated seedlings were used. The occupancy is displayed as log 2 fold change and was calculated as the ratio between the respective ChIP and the respective IgG control in 100 bp bins from 2.4 kb upstream to 7.7 kb downstream of the transcriptional start site (TSS) of EIN2 . Occupancies that were lower in the ChIP sample compared to the IgG sample were set to zero. The region with significant EEN enrichment determined with SICER is indicated as well. ( B ) FLAG - pull-down assays using FLAG-tagged EEN and Halo-tagged ARP5 that were in vitro translated in rabbit reticulocyte extract. Anti-FLAG and anti-Halo antibodies were used to confirm EEN/ARP5 expression, EEN immunoprecipitation and EEN-ARP5 interaction. ( C ) Heatmap illustrates the log 2 fold change in mRNA expression of INO80-dependent genes in untreated 3-day-old etiolated ino80-8 , een-2 and arp5-1 seedlings relative to their expression in Col-0 seedlings. INO80-dependent genes were selected as genes that show a significant differential expression in ino80-8 mutant seedlings compared to Col-0 seedlings (787 genes up, 1079 genes down). ( D ) Genome browser screenshot shows the occupancy of H3K27me3 (right) and H2A.Z (left) at the EIN2 gene in untreated 3-day-old etiolated Col-0, ref6-1 , een-2, ref6-1 een-2, ino80-1, ref6-1 ino80-1, arp5-1 and ref6-1 arp5-1 seedlings. Genome-wide occupancy of H3K27me3 and H2A.Z was determined by ChIP-seq. To ensure an accurate comparison of individual chromatin features between genotypes, the tracks were normalized to the respective sequencing depth. Normalization was separately done for each chromatin feature.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: ChIP-sequencing, Sequencing, Control, In Vitro, Expressing, Immunoprecipitation, Quantitative Proteomics, Mutagenesis, Genome Wide, Comparison

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) ( B ) Aggregated H2A.Z profile of all Arabidopsis genes (TAIR10) in L er , ein6-1 , een-1 , ein6-1 een-1 ( A ) and Col-0, ref6-1 , een-2, ref6-1 een-2, ino80-1, ref6-1 ino80-1 , arp5-1 and ref6-1 arp5-1 ( B ) seedlings from 1 kb upstream to 2 kb downstream of the TSS determined by ChIP-seq. ( C ) ( D ) ET-induced H2A.Z eviction dynamics are shown as aggregated H2A.Z profiles of 3-day-old etiolated L er , een-1 , Col-0 and ino80-8 seedlings treated for 4 hr with ET gas. Two biological H2A.Z ChIP-seq replicates for L er and een-1 ( C ) and one for Col-0 and ino80-8 ( D ) were analyzed. Profiles visualize the log 2 fold change between the respective air and ET-treated H2A.Z ChIP-seq samples from 1 kb upstream to 2 kb downstream of the TSS. 1076 genes that show a robust H2A.Z eviction in response to ET in all replicates (≥1.3 fold SICER comparison, air vs 4 hr ET) are shown as solid lines whereas all genes (TAIR10) are shown as dashed lines.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: ChIP-sequencing, Comparison

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Aggregated EEN and H2A.Z profile of the 1000 top-ranked genes that show EEN binding from 1 kb upstream to 2 kb downstream of the TSS. Two independent EEN ChIP-seq datasets (Col-0 35S:EEN ) as well as one Col-0 H2A.Z ChIP-seq dataset (Col-0) is shown. ( B ) Pie chart illustrates the genomic distribution of EEN binding sites. ( C ) Gene ontology enrichment analysis of the 1000 top-ranked EEN targets using the functional annotation tool DAVID. ( D ) Heatmaps show ET-induced H2A.Z eviction dynamics in 3-day-old etiolated L er , een-1 , Col-0 and ino80-8 seedlings treated for 4 hr with ET gas. The heatmaps correspond to the aggregate profiles in . Heatmaps visualize the log 2 fold change between the respective air and ET-treated H2A.Z ChIP-seq samples from 1 kb upstream to 2 kb downstream of the TSS. (ET) indicates that the dynamics of 1076 genes with a robust H2A.Z eviction in response to ET in all replicates (≥1.3 fold SICER comparison, air vs 4 hr ET) is shown whereas (all) indicates that the dynamics of all Arabidopsis genes is shown.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: Binding Assay, ChIP-sequencing, Functional Assay, Comparison

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet: ( A ) Genome browser screenshot visualizes levels of H3K27me3 at the EIN2 gene in the indicated tissues of L er and ein6-1 een-1 plants (dry seeds, roots and shoots of etiolated seedlings, de-etiolated whole-seedlings, flowers and siliques). H3K27me3 occupancy was identified with ChIP-seq. The tracks were normalized to the respective sequencing depth for each experiment to allow an accurate comparison between L er and ein6-1 een-1 H3K27me3 profiles. Normalization was separately done for each chromatin feature. ( B ) Histochemical GUS staining of untreated three-day-old etiolated L er and ein6-1 een-1 seedlings stably expressing either a EIN2 promoter GUS fusions with ( EIN2:GUS ) or without the 5’UTR intron ( EIN2ΔI:GUS ). Schematic illustration of the used EIN2:GUS constructs is shown as well. The gray box indicates the 2 kb promoter region of EIN2 , the green boxes represent the 5’UTR and the black line indicates the 5’UTR intron. ( C ) Hypothetic model of the EIN6 (REF6)/INO80-mediated double safeguard mechanism at the 5’UTR intron of EIN2 . Besides the demethylase function role of EIN6 (REF6) in antagonizing PRC2-mediated tri-methylation of H3K27, we speculate that EIN6 (REF6) also antagonizes SWR1-facillitated H2A.Z incorporation at EIN2 . This scenario of a dual function holds true for the INO80 complex as well which counteracts SWR1 by removing H2A.Z, but also antagonizes H3K27 trimethylation through an unknown mechanism. The resulting low levels of H3K27me3 and H2A.Z possibly increase the accessibility of the enhancer element for binding of yet unknown transcription factors (TFs). When this EIN6 (REF6)/INO80-mediated regulation is functionally impaired, H3K27me3 and H2A.Z start to accumulate over the entire gene body of EIN2 mutually reinforcing their depositions.

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: ChIP-sequencing, Sequencing, Comparison, Staining, Stable Transfection, Expressing, Construct, Methylation, Binding Assay

Journal: eLife

Article Title: Epigenetic silencing of a multifunctional plant stress regulator

doi: 10.7554/eLife.47835

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-ubiquityl-Histone H2A (Lys119) , Cell Signaling Technology , Cat#: 8240 RRID: AB_10891618 , 10 μl Materials and methods subsection: ChIP-seq.

Techniques: Transformation Assay, Western Blot, In Vitro

Journal: Frontiers in Immunology

Article Title: m 6 A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection

doi: 10.3389/fimmu.2021.705232

Figure Lengend Snippet: Downregulation of Alkbh5 and Fto in intestinal epithelial cells C. parvum infection. (A) Heatmaps showing expression profile of key genes involved in the m 6 A RNA methylation machinery in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24 h followed by genome-wide array analysis. (B) Dynamics of Alkbh5 and Fto downregulation in IEC4.1 cells following C. parvum infection. IEC4.1 were exposed to C. parvum infection for 4-48 h and RNA expression levels of Alkbh5 and Fto were validated by using real-time quantitative PCR. Expression levels of Cxcl2 (as a positive control), Mettl3, Mettl14 and Wtap were also measured. (C) Decreased abundance of Alkbh5 and Fto proteins in IEC4.1 cells following C. parvum infection. IEC4.1 were exposed to C. parvum infection for 48-72 h and expression levels of Alkbh5 and Fto at the protein level were validated using Western blot. β-Actin was also blotted for internal control. Representative gels were shown. (D) Downregulation of Alkbh5 and Fto in murine intestinal epithelium following C. parvum infection in vivo . Neonates of mice at 5 days of age received C. parvum administration by oral gavage and intestinal ileum epithelium were isolated after infection for 24h. Expression levels of expression levels of Alkbh5 and Fto were measured. (E) Downregulation of Alkbh5 and Fto in 2D murine intestinal epithelial monolayers following C. parvum infection ex vivo . Data represent three independent experiments. *p<.05 vs the non-infected control.

Article Snippet: The mouse Alkbh5 siRNA (#sc-141022) and human ALKBH5 siRNA (#sc-93856) were purchased from the Santa Cruz Biotechnology.

Techniques: Infection, Expressing, Methylation, Genome Wide, RNA Expression, Real-time Polymerase Chain Reaction, Positive Control, Western Blot, Control, In Vivo, Isolation, Ex Vivo

Journal: Frontiers in Immunology

Article Title: m 6 A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection

doi: 10.3389/fimmu.2021.705232

Figure Lengend Snippet: C. parvum infection causes downregulation of Alkbh5 and Fto with the involvement of NF-кB signaling activation. (A) Downregulation of Alkbh5 in IEC4.1 cells following C. parvum infection is MyD88-dependent. Knockout MyD88 in IEC4.1 cells blocked the suppression of Alkbh5 induced by C. parvum . Cxcl2 induction in cells in response to infection was also measured for positive control. (B) Luciferase activity associated with the promoters of both Alkbh5 and Fto genes in IEC4.1 cells following C. parvum infection. Cells were transfected with the generated reporter constructs and then exposed to C. parvum infection for 12h, followed by measurement of luciferase activity. Cells transfected with the empty vector were used as control. (C) Luciferase activity associated with the promoter of Alkbh5 in MyD88-/- IEC4.1 cells following C. parvum infection or TNF-α stimulation. IEC4.1 cells deficient in Myd88 were exposed to C. parvum infection (for 12h) or TNF-α stimulation (for 6h). Luciferase activity was measured. (D) Recruitment of NF-кB p65 to the Alkbh5 promoter region in IEC4.1 cells following C. parvum infection or TNF-α stimulation. Cells were exposed to C. parvum infection (for 24 h) or TNF-α stimulation (for 4h), followed by ChIP analysis using anti-p65 and the PCR primer sets as designed. Increased recruitment of p65 was detected in the -1571~-1361 (Set-1) region of the Alkbh5 gene locus in cells following C. parvum infection or TNF-α stimulation. (E) Recruitment of NF-кB subunits and HDAC1, as well as enrichment of H3K9me3 and H3K27me3, at the Alkbh5 promoter region in intestinal epithelial cells following C. parvum infection. Cells were exposed to C. parvum infection for 24 h, followed by ChIP analysis using anti-p65, anti-p50, anti-HDAC1, anti-H3K9me3, or anti-H3K27me3 and the PCR primer Set-1. Recruitment of NF-кB subunits and HDAC1, as well as enrichment of H3K9me3 and H3K27me3, at the ApoE promoter region in cells following C. parvum infection were also measured as a positive control. Data represent three independent experiments. *p<.05 vs the non-infected control.

Article Snippet: The mouse Alkbh5 siRNA (#sc-141022) and human ALKBH5 siRNA (#sc-93856) were purchased from the Santa Cruz Biotechnology.

Techniques: Infection, Activation Assay, Knock-Out, Positive Control, Luciferase, Activity Assay, Transfection, Generated, Construct, Plasmid Preparation, Control

Journal: Frontiers in Immunology

Article Title: m 6 A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection

doi: 10.3389/fimmu.2021.705232

Figure Lengend Snippet: m 6 A methylation modulates intestinal epithelial innate defense against C. parvum infection. (A) Knockdown of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for Alkbh5 or Fto and the HDR plasmids. Stable transfected cells were cloned and confirmed by real-time PCR and Western blot analysis. Gapdh was also blotted for control. (B) Knockdown of Alkbh5 or Fto in IEC4.1 cells decreased the infection burden of C. parvum infection. Knockdown of Alkbh5 or Fto increased m 6 A RNA methylation in IEC4.1 cells, as measured by m6A RNA methylation quantitation assay and dot blot. IEC4.1 cells and cells deficient with Alkbh5 or Fto were then exposed to C. parvum infection for 24 h. IEC4.1 cells transfected with the empty vector, marked as IEC4.1-WT (wild type) were used as the control. Infection burden of C. parvum was quantified by measuring parasite cpHsp70 or cp18s using real-time PCR. (C) Knock-in of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for active Alkbh5 (Alkbh5-KI) or Fto (Fto-KI) vectors. Stable transfected cells were cloned and confirmed by Western blot analysis. (D) Overexpression of Alkbh5, but not Fto, increased the infection burden of C. parvum in IEC4.1 cells. Cells stably expressing Alkbh5 or Fto were exposed to C. parvum infection for 24 h and infection burden of C. parvum was quantified. (E) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in IEC4.1 cells. Cells were treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. (F) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in 2D intestinal epithelial monolayers. Monolayers were cultured and treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. Data represent three independent experiments. *p<.05 vs cells transfected with the empty-vector control (as IEC4.1-WT in A, B, D ) or cells treated with the control-siRNA (in E, F ).

Article Snippet: The mouse Alkbh5 siRNA (#sc-141022) and human ALKBH5 siRNA (#sc-93856) were purchased from the Santa Cruz Biotechnology.

Techniques: Methylation, Infection, Knockdown, Transfection, CRISPR, Clone Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Quantitation Assay, Dot Blot, Plasmid Preparation, Knock-In, Over Expression, Stable Transfection, Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: m 6 A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection

doi: 10.3389/fimmu.2021.705232

Figure Lengend Snippet: Expression levels of Irgm2 and Igtp are increased and with a decreased m 6 A methylation in IEC4.1 cells following C. parvum infection. (A) Increased expression levels of both Irgm2 and Igtp genes with a decreased m 6 A peaks in their mRNAs in IEC4.1 cells following C. parvum infection. RNA levels of Irgm2 and Igtp and their m 6 A levels were assessed by RNA-Seq and m 6 A-RIP-Seq, respectively. (B) Decrease in their m 6 A levels in cells following C. parvum infection occurred in the promoters/UTRs/CDS regions. (C) Increased RNA stability of Irgm2 mRNA in Alkbh5-/- IEC4.1 cells versus that in IEC4.1 cells. Data represent three independent experiments. *p<.05 vs the non-infected control or cells transfected with the empty-vector control (as IEC4.1-WT in C ).

Article Snippet: The mouse Alkbh5 siRNA (#sc-141022) and human ALKBH5 siRNA (#sc-93856) were purchased from the Santa Cruz Biotechnology.

Techniques: Expressing, Methylation, Infection, RNA Sequencing, Control, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: m 6 A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection

doi: 10.3389/fimmu.2021.705232

Figure Lengend Snippet: m 6 A methylation-mediated intestinal epithelial anti- C. parvum defense in human intestinal epithelium. (A) Increase of global m 6 A RNA methylation in HCT-8 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24h and m 6 A RNA methylation was measured by m 6 A RNA methylation quantitation assay. (B) Decrease of ALKBH5 and FTO expression levels in HCT-8 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24h and expression levels of ALKBH5 and FTO was measured by real-time PCR. (C) Knockdown of ALKBH5 via siRNA in HCT-8 cells. Cells were treated with the siRNA to ALKBH5 for 24h and knockdown of ALKBH5 was confirmed by real-time PCR. Cells transfected with non-specific control siRNA were used as the control. (D) Knockdown ALKBH5 in HCT-8 cells decreased C. parvum infection burden. HCT-8 cells were first treated with the siRNA-ALKBH5 and then exposed to C. parvum infection for 24h. Infection burden of C. parvum was quantified by measuring parasite cpHsp70 or cp18s using real-time PCR. Data represent three independent experiments. *p<.05 vs the non-infected control (in A–D ).

Article Snippet: The mouse Alkbh5 siRNA (#sc-141022) and human ALKBH5 siRNA (#sc-93856) were purchased from the Santa Cruz Biotechnology.

Techniques: Methylation, Infection, Quantitation Assay, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Transfection, Control

Journal: Cancer gene therapy

Article Title: Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

doi: 10.1038/cgt.2016.8

Figure Lengend Snippet: Figure 3. Expression of calreticulin (CRT) on the cell surface. SCCVII cells were infected with RH2 at a multiplicity of infection (MOI) of 10, incubated for 24 h, and fixed in paraformaldehyde. Cells were stained with an anti-CRT antibody, Alexa Fluor 633-conjugated wheat germ agglutinin (WGA), and (4',6-diamidino-2-phenylindole) (DAPI) and then analyzed under a confocal laser-scanning microscope. A representative result was presented. PBS, phosphate-buffered saline; SCC, squamous cell carcinoma.

Article Snippet: After washing, the cells were incubated with an FITC (fluorescein isothiocyanate)-conjugated goat polyclonal antibody diluted 1:100 and Alexa Fluor 633-conjugated wheat germ agglutinin (WGA) (Invitrogen, Carlsbad, CA, USA) for 30 min. After washing, coverslips were mounted onto microslides using a ProLong Gold Antifade Reagent with DAPI (4',6-diamidino-2-phenylindole) (CST Japan, Tokyo, Japan).

Techniques: Expressing, Infection, Incubation, Staining, Laser-Scanning Microscopy, Saline