Journal: Nature communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis.

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: Fig. 2 Single-cell RNA-seq identified subpopulations of cardiomyocytes. a Late stage cardiomyocytes (4,689 cells with TNNT2 and ACTC1 expression from day 14 and day 45) were further resolved using ICGS to identify subpopulations of cardiomyocytes. Associated t-SNE cell populations were colored by day of differentiation, as well as by cluster identified (markers within each cluster beside each cluster). b Six cardiomyocyte populations were identified representing subpopulations of cardiomyocytes in early proliferative stages (Cluster 4, 5) expressing cyclins, mid-cardiomyocyte stage (Cluster 2, 3) expressing HAND1, and more mature cardiomyocytes (Cluster 0, 1) expressing sarcomeric (MYH6, MYL2, TNNT2, MYBPC3) and calcium handling genes (RYR2, PLN). c Single cells from day 30 of differentiation were profiled using an independent technology (Fluidigm C1) to resolve coincident mid-to-late state differentiation heterogeneity. Genes associated with distinct observed cell populations identified by the algorithm MarkerFinder are shown. Factors with defining expression in each of the HOPACH clustered populations are shown (SF1, ISL1, JARID2, TBX5, MYL2/HEY2, and HOPX). Two major groups in hiPSC-CMs were observed to express either NR2F2 or MYL2/HEY2. Within each group, identified heart developmental regulators were identified using the software GO-Elite (left panel)

Article Snippet: To overexpress NR2F2, TBX5, and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: RNA Sequencing, Expressing, Software

Journal: bioRxiv

Article Title: Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

doi: 10.1101/678185

Figure Lengend Snippet: A) Wholemount in situ hybridization comparing mmp2, mmp14a and mmp14b expression in WT vs pax2a −/− and DMSO vs DMH4 treated embryos at 32hpf. Mmp2, 14a and 14b signal within the optic fissure (yellow arrowhead) appears decreased in pax2a −/− and DMH4 treated embryos. B) qPCR analysis confirms a decrease in expression of mmp2, 14a and 14b in both pax2a −/− and DMH4 treated embryos. C) Two color wholemount in situ hybridization simultaneously examining mmp2 and kdrl , or mmp2 and rorB expression at 32hpf. DNA was stained with DAPI. Scale bar = 50μm. Clear overlap of signal is observed for mmp2 and kdrl , but not mmp2 and rorB . D) Whole mount Immunohistochemistry was used to visualize laminin (red) in ARP101 treated Tg[ rx3 :GFP] embryos at 48hpf. Central-proximal regions of the optic fissure are displayed. E) Quantification of 48hpf ARP101 treated embryos for fissure fusion (absence of laminin signal), partial fusion (partial retention of laminin) or failure to fuse (retention of laminin throughout the fissure). n= 41 (10mM), 38 (15mM), 42 (20mM) ANOVA p<0.0001. F) Quantification of 48hpf ARP101 treated embryos for fissure fusion, partial fusion or failure to fuse after various treatment initiation times from 24-32hpf. n= 42 (24-48hpf), 29 (26-48hpf), 29 (28-48hpf), 23 (30-48hpf), 37 (32-48hpf). ANOVA p<0.0001.

Article Snippet: For ARP101 (Tocris) treatments embryos were dechorionated and treated with 10, 15, 20, 25 or 30μM ARP101 in fish water from 24 to 48hpf, unless stated otherwise.

Techniques: In Situ Hybridization, Expressing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

doi: 10.1101/678185

Figure Lengend Snippet: A) Brightfield images of DMSO and ARP treated embryos at 48hpf. Concentrations of ARP101 exceeding 20uM result in toxic effects. B) Whole mount Immunohistochemistry was used to visualize laminin (red) in ARP101 treated Tg[ rx3 :GFP] embryos at 48hpf. Central-proximal and distal regions of the optic fissure are displayed.

Article Snippet: For ARP101 (Tocris) treatments embryos were dechorionated and treated with 10, 15, 20, 25 or 30μM ARP101 in fish water from 24 to 48hpf, unless stated otherwise.

Techniques: Immunohistochemistry

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 1. Salient characteristics of C>U RNA editing by A3G in 293T cells. (a) Mean and range of editing level at the 712 sites identified as targets for A3G-mediated editing are shown for the three A3G transfectant samples. The sites are ordered by the mean editing level. (b) Logo indicating sequence conservation and base frequency for sequences bearing the editing sites (at position 0). (c) Histogram of nucleotide lengths (b) of inverted repeat sequences flanking the editing sites.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Transfection, Sequencing

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 2. Transient overexpression of A3G induces C>U RNA editing in 293T transfectants. Sanger chromatograms of cDNAs (in duplicate) and genomic DNAs (gDNA) (in triplicate) from control and A3G transfectants. Edited C is shaded black.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Over Expression, Control

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 3. Site-specific C>U RNA editing in mRNAs co-purified with rA3G. (a) Immunoblot showing WT A3G expressed in whole cell lysate (20 μg) of 293T cells and recombinant (r) WT A3G (500 ng) obtained from 293T cells (purchased from Origene, Rockville, MD). Full-length blot is presented in Supplementary Fig. S4 (b) Cytidine deamination activity of recombinant A3G was examined in an in vitro reaction with a 5ʹ fluorescent dye-labeled ssDNA substrate of 40 nucleotides (left panel). Full-length gel is presented in Supplementary Fig. S4. Sanger chromatograms of cDNAs of MED1 RNA from in vitro RNA editing assay containing rA3G in the presence of 100 nM (+) or 1 μM (++) and ART MED1 RNA (right panel). (c) Sanger chromatograms of cDNAs of A3G substrates (MED1, KIAA1715, SCD, ITFG1, RFX7, GOLGA5, CHMP4B, CLASP1) (left panel) and A3A substrates (VIM, ASCC2, TMEM179B, SDHB) (right panel) from in vitro RNA editing assay containing only rA3G or from in vitro RNA editing assay containing in vitro transcribed SDHB RNA and purified A3A protein (SDHB lower panel). Edited cytidines are highlighted in black.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Purification, Western Blot, Recombinant, Activity Assay, In Vitro, Labeling

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 4. Site-directed mutagenesis of A3G shows requirement for both N- and C–terminal domain zinc dependent deaminase motif residues for site-specific RNA editing. (a) Chromatograms of cDNAs (single) from ctrl./A3G transfected 293T cells shows the effect of mutations in A3G-NTD (W94A, C97S, W127A) and A3G-CTD (C291S) on C>U RNA editing (edited C is shaded black) in selected genes. (b) Bar graph showing RNA editing level of GOLGA5, KIAA1715 and MED1 in A3G-NTD and -CTD mutant transfectants. RNA editing levels (ratio of edited versus total RNA) are calculated by SequencherTM software. The detection limit for relative height of minor peaks was 4.86% (depicted by a dotted line). Mean and SEM (n = 3) (c) Immunoblot showing A3G protein expression in whole cell lysates of 293T cells when transfected with empty vector (control), WT or various A3G-NTD and -CTD mutants. The D128K and P129A mutants were run on separate gels on the same day. The dashed line separates the two gels. Full-length blot is presented in Supplementary Fig. S4 (d) Bar graph depicting RNA editing level of KIAA1715, PRPSAP2, SCD and TM7SF3 cDNAs in WT or mutant transfectants. The detection limit is depicted by a dotted line. Mean and SEM (n = 3). Statistical analysis was performed by one- way ANOVA followed by multiple comparisons of RNA editing levels between WT and the mutants. Editing level is considered 0%, when the Sequencher software detects no secondary T peak. ****=adjusted P value ≤ 0.0001; NS = Not Significant.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Mutagenesis, Transfection, Software, Western Blot, Expressing, Plasmid Preparation, Control

Journal: PLoS Pathogens

Article Title: Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-Cells

doi: 10.1371/journal.ppat.1000342

Figure Lengend Snippet: (A) Quiescent CD4+ T-cells were purified from freshly isolated PBMCs of two independent donors with CD4 magnetic beads and nucleofected with siRNA targeting APOBEC3G (siA3G 883 ), conjugated with (FITC) or without FITC (None). Cells were analyzed by flow cytometry 3 hr after nucleofection. (B) To monitor the integrity of the RNAi machinery, quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNA targeting CD4 (siCD4), control siRNA (siControl), or no siRNA (None). The levels of cell surface CD4 expression were monitored by flow cytometry using PE-conjugated anti-CD4 antibody or isotype-matched control 48 hr after nucleofection and represented by mean fluorescent intensity (MFI) in each panel. The solid line represents PE-CD4 antibody stained cells, whereas the shaded area represents isotype control staining.

Article Snippet: After blocking with 5% skim milk in PBS with 0.05% Tween-20 (PBS-T), membranes were reacted with either polyclonal anti-APOBEC3G antibody (Cat. No. 10201, NIH AIDS Research and Reference Reagent Program) or polyclonal anti-β-Actin (Rockland Immunochemicals Inc., Gilbertsville, PA).

Techniques: Purification, Isolation, Magnetic Beads, Flow Cytometry, Derivative Assay, Control, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-Cells

doi: 10.1371/journal.ppat.1000342

Figure Lengend Snippet: (A) Quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNAs and cultured for two days. Total RNA was isolated, and the levels of APOBEC3G mRNA were monitored by quantitative real time RT-PCR using β-actin as an internal control . P values (asterisks) versus control siRNA were 0.00022 (siA3G 240 WT ), 0.00013 (siA3G 726 ), 0.00005 (siA3G 883 ), respectively. (B) Quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNAs and cultured for two days. Cells were lysed in 0.5% SDS, and the levels of APOBEC3G protein were monitored by Western blotting. β-actin was used as a loading control.

Article Snippet: After blocking with 5% skim milk in PBS with 0.05% Tween-20 (PBS-T), membranes were reacted with either polyclonal anti-APOBEC3G antibody (Cat. No. 10201, NIH AIDS Research and Reference Reagent Program) or polyclonal anti-β-Actin (Rockland Immunochemicals Inc., Gilbertsville, PA).

Techniques: Derivative Assay, Cell Culture, Isolation, Quantitative RT-PCR, Control, Western Blot

Journal: Nature communications

Article Title: An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

doi: 10.1038/s41467-023-36967-2

Figure Lengend Snippet: Fig. 2 | NR2F2-Iso2 promotes anchorage-independent growth and melanoma sphere formation in vitro. a qRT-PCR showing relative NR2F2-Iso1 and NR2F2-Iso2 transcript levels normalized to SK-MEL-147, where NR2F2-Iso2 is partly methylated (M) and unmethylated (U). Methylation status was determined with Illumina 450 K arrays and/or methylation specific PCR. b Western blots of melanoma cell lines probed with Isoform-specific NR2F2 antibodies. c Heatmap illustrating CpG- methylation status of NR2F2-Iso2 based on Illumina 450 K array data. d Pie charts showing % CpG methylation of indicated sites at the NR2F2 locus as determined by bisulfite sequencing. Asterisks denote CpGs interrogated in 450 K arrays. e qRT- PCR (left) and western blotting (right) show that treatment of MeWo cells with the demethylating agent 5-aza-2′-deoxycytidine (2.5 μM aza, 72 h) permits NR2F2-Iso2 expression (bars represent SD). One experiment of 2 biological replicates is shown; further validated in other cell lines in Supplementary Fig. 7. f qRT-PCR and western blotting validate isoform-specific NR2F2-Iso2 depletion in NR2F2-Iso2 expressing 4L cells with shNR2F2-Iso2 (shA, shB) compared to shSCR (control) (bars represent SD). Actin served as loading control. Representative result from one of multiple repeats is shown (>3). g Growth rates of shSCR and shNR2F2-Iso2 expressing 4L

Article Snippet: Lentiviral vectors were also used for NR2F2 ectopic expression: NR2F2 Isoform 1 (EX-C0221-Lv205, GeneCopoeia) and NR2F2 Isoform 2 (RC226609L2, Origene). pEZLv205 (EX-NEG-Lv205, GeneCopoeia) or pLenti-C-mGFP (PS100071, Origene) were used as control vectors.

Techniques: In Vitro, Quantitative RT-PCR, Methylation, Western Blot, CpG Methylation Assay, Sequencing, Expressing, Control

Journal: Nature communications

Article Title: An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

doi: 10.1038/s41467-023-36967-2

Figure Lengend Snippet: Fig. 6 | NR2F2-Iso1 reduces colony formation ability and metastatic potential. a qRT-PCRand b western blotting confirms ectopic NR2F2-Iso1 expression in MeWo cells (bars represent SD). c Bar graph showing significantly reduced colony forming ability of MeWo cells that ectopically express NR2F2-Iso1 28 days after seeding (two-tailed unpaired T test, bars represent SD). d qRT-PCR data and e western blotting showing differences in NR2F2-Iso1 expression between shSCR and shNR2F2-Iso1 (shX, shY) MeWo cells that ectopically express GFP or GFP-NR2F2- Iso2 (bars represent SD). f Bar graphs showing relative colony forming efficiencies of these cells 21 days after seeding (two-way ANOVA, bars represent SD) (n = 6 experimental replicates). Experiments in a–f have been done al least twice; c and f three times. (g–i) 4L cells (n = 9 GFP, 10 Iso1 OE NSG mice) labeled with lentiviruses

Article Snippet: Lentiviral vectors were also used for NR2F2 ectopic expression: NR2F2 Isoform 1 (EX-C0221-Lv205, GeneCopoeia) and NR2F2 Isoform 2 (RC226609L2, Origene). pEZLv205 (EX-NEG-Lv205, GeneCopoeia) or pLenti-C-mGFP (PS100071, Origene) were used as control vectors.

Techniques: Western Blot, Expressing, Two Tailed Test, Quantitative RT-PCR, Labeling