Journal: Nature Communications

Article Title: Compartmentalized cytoplasmic tradewinds direct soluble proteins

doi: 10.1038/s41467-026-70688-6

Figure Lengend Snippet: a Asymmetry analysis of spot activation of mEos3.2 Arp3, vinculin, and paxillin in CAD cells illustrates that they are targeted toward the leading edge, where polymerization and adhesions initiate ( n = 14, Arp3, 9 vinculin, 10 paxillin cells). b Temporal color-coded spot activation of mEos3.2 actin in an NG108 cell advancing preferentially on the right illustrates that this is the region where recently activated actin is targeted (arrows). c Maximum projection of 3D SIM images of Alexa 647-phalloidin and mEmerald MLC in control cells and cells treated with 0.2 μM nitro-blebbistatin before fixation illustrates the broadening and flattening of the actin-myosin arcs at the border between the lamella and cell body when myosin is inhibited. d These observations were quantified by fitting the arcs to ellipses and calculating ellipticity, which reveals flatter arcs in the blebbistatin-treated cells ( n = 19 control cells, 87 measurements, 17 blebbistatin cells, 100 measurements). Ellipticity =√(1-(minor) 2 major −2 )) e An overlay of two different mNeon MLC image time points ( t = 0, and t = 45 s) with arrows indicating where the edge retracts and extends during the 45 s. f Contour of the same two-time points in ( e ) with arrows indicating changes in the position and curvature of the arcs corresponding to the retraction and advancement of the leading edge. g CAD cell expressing mEmerald MLC were treated with a high-power localized beam of 405 light positioned over one arc, causing the arc to be disrupted and only the leading edge in front of that single arc to collapse. The yellow line indicates the position of the edge prior to MLC disruption. p values two-tailed t -tests. Box plots show median (center line), interquartile range (25th–75th percentiles; box), whiskers to the most extreme non-outlier values, and notches as 95% CIs. Scale bars, 5 μm. Source data are provided as a Source Data file.

Article Snippet: Antibodies used were: anti-actin (Sigma, A5441), anti-paxillin (Sigma, P1093), GFP (Abcam, AB38689), GAPDH (Applied Bioscience, AM4300), Eos (Novus, NP3-05557), Arp3 (Proteintech, 13822), and Vinculin (Sigma, V9131).

Techniques: Activation Assay, Control, Expressing, Disruption, Two Tailed Test

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR10 does not affect actin nucleation. a Immunoblots lysates of HL-60-derived neutrophils (2 × 10 6 ). TLR10 gene knockdown was performed as above and cells were stimulated with LPS (1 μg/mL) for 60 min. Actin nucleation proteins ARP3 (1:500) and Diap1 (1:400) were detected in the blot corresponding to the specific molecular weight. Molecular weight is depicted on the left side of the blots. β-Actin showed in the lower panel referred as loading control. b , c Densitometry analysis showed no significant change in actin nucleation proteins.

Article Snippet: Immunoblots were prepared by anti-TLR10 antibody (1:600, 1 mg/mL; Abcam, Cambridge, MA, USA), anti-Diap1 antibody (1:500, Cell Signaling Tech, Danvers, MA, USA), anti-ARP3 antibody (1:400, Cell Signaling Tech, Danvers, MA, USA).

Techniques: Western Blot, Derivative Assay, Knockdown, Molecular Weight, Control

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a-c Arp3 actin comet tail in Lm EGD infected MEFs at 6 h post infection (MOI 10). Bacteria are shown in green, and Arp3 in magenta. Fluorescent intensity profile (FIP) was taken of the dotted line along the midline of the cell in the inset image WT ( a ), USP18 C61A/C61A ( b ), and isg15 −/− ( c ), scale bar, 1 μm. d Percentage of bacteria with Arp3 surface localization at 6 HPI, representative data (WT n=5, CA n=9, KO n=10) analyzed with Kruskal-Wallis test followed by Dunn’s post hoc test. e Stimulated Emission Depletion (STED) images of bacteria in MEFs at 6 h post infection, bacteria are shown in green (a-ActA), and actin (Phalloidin) in magenta. scale bar, 1 μm. f Data from machine learning analysis of average length of actin comet tails formed by bacteria at 6 h post infection in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs analyzed by Kruskal-Wallis test with Dunn’s post hoc. g Average thickness of actin comet tails formed by bacteria at 6 h post infection in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs, compared by by Kruskal-Wallis test with Dunn’s post hoc. h Average signal intensity of actin structures along bacterial actin tails in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs analyzed by Kruskal-Wallis test with Dunn’s post hoc. In f-g bar graphs represent the mean ± SEM and results analyzed from three independent experiments WT n=439, CA n=661, KO n=680. i Representative images of Lm actin comet tail formed at 24 HPI using STED imaging, bacteria in green (a-ActA), and actin (Phalloidin) in magenta; scale bar, 5 μm. j Wildtype, USP18 C61A/C61A , and isg15 −/− MEFs infected with Western Reserve strain of Vaccinia virus (VACV) for 8 h. Actin is shown in magenta, and VACV in green; scale bar,10 μm, insets, 1 μm. k Average length of VACV actin tail in MEFs; data represent the mean ± SEM, WT n=1183, CA n=3286, KO n=1752, significance determined by Kruskal-Wallis test with Dunn’s post hoc. l Representative confocal images of VACV actin comet tails at 12 HPI in WT and USP18 C61A/C61A using anti-anti-VACV 14K antibody (green) and phalloidin for actin (magenta); scale bar, 10 μm, insets, 1 μm. In d , f , g , h and k asterisks indicate p values with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Infection, Imaging, Western Blot

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Time-lapse microscopy of Lm GFP-EGD (green) infected mTagRFP-T-lifeact-7 (magenta) expressing wildtype, USP18 C61A/C61A , or isg15 −/− MEFs at 4 h post infection imaged every 10s. Scale bar, 5 μm. SI movies 1-5. b Quantification of average speed per bacterium tracked from 3 independent experiments (WT n=61, CA n=35, KO n=75), analyzed by Kruskal-Wallis test with Dunn’s post hoc. c-e Model of ISG15 (in rose) C terminus attached to Lys18, Lys75, and Lys191 of Actin-related protein 3 (Arp3) in the inactive Arp2/3 complex PDB ID: 6YW7 ( c ); the active Arp2/3 complex PDB ID: 7AQK ( d ); the active Arp2/3 complex with branched actin filaments PDB: 7AQK ( e ). f Schematic of ActA domains with interacting proteins; inset amino acid sequence mutated to abrogate Arp2/3 recruitment and comparison of R148S with WT for motility and autophagy; created with Biorender.com. g Quantification of average speed of motility per bacterium of wildtype bacteria and Lm EGD DactA complemented with ActA R148S which unstably recruits the Arp2/3 complex tracked from three independent experiments (WT n=191, CA n=203, KO n=164) by Brown-Forsythe ANOVA test with Dunnett’s T3’s post hoc. h Representative time-lapse images of Lm EGD DactA ::ActA R148S in mTagRFP-T-lifeact-7 (magenta) expressing wildtype, USP18 C61A/C61A , or isg15 −/− MEF at 4 h post infection imaged every 10s. Scale bar, 5 μm. White arrows point to inconsistent actin tails. Yellow arrows highlight normal consistent bacterial actin tails. See also SI movies 6-8. i Representative Lm 10403s plaques in USP18 C61A/C61A mCherry-Arp3 and mCherry-Arp3 3KR at 3 DPI. j Plaque size (in square pixels) from 3 independent experiments (WT n=1073, KR n=768), analyzed with Kolmogorov-Smirnov’s t-test. In b, g, i and j asterisks indicate p values with **p < 0.01, ***p < 0.001, and ****p<0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Time-lapse Microscopy, Infection, Expressing, Sequencing

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Validation of ActA expression in Lm wild-type EGD, EGD ΔactA , and EGD ΔactA strain complemented with wildtype ActA, ActA R148S, or ActA R148S GFP constructs. b Representative image of plaque formation of EGD ΔactA::actA R148S infection (MOI 0.001) in WT, USP18C64A and isg15 −/− A549 at 4 DPI. c Plaque size (in square pixels) from four independent experiments (WT n=742, CA n=945, KO n=653), analyzed with one-way ANOVA followed by Tukey’s post hoc (*p < 0.05, and **p < 0.01, raw data are available in the source data file). d Validation of deletion of endogenous Arp3 in usp18 C61A/C61A MEFs. usp18 C61A/C61A actr3 −/− MEF clone was complemented with mCherry-Arp3 wildtype or mCherry-Arp3 that harbors lysine mutations to arginine at Lys18, Lys75, Lys191. e Representative image of plaque formation of Lm 10403s in USP18 C61A mCherry-Arp3 and mCherry 3KR Arp3 (K18/75/191R) at 3 DPI. Infected cell monolayers were stained with a-InlC (black) and crystal violet (pink). f Immunoprecipitation of mCherry tag in mCherry-Arp3 complemented usp18 C61A/C61A actr3 −/− MEFs treated with Type I IFN (1000 units/mL) for 24 h; SDS-PAGE for both Arp3 and ISG15. The experiment was repeated three times, and one representative blot is shown. g Immunoprecipitation of mCherry in mCherry-Arp3 complemented usp18 C61A/C61A actr3 −/− MEFs infected with Lm EGD (MOI 10) for 24 h; SDS-PAGE for both Arp3 and ISG15. Blot from one representative experiment is shown. In f and g , asterisks indicate background bands from strip and reprobe.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Expressing, Construct, Infection, Staining, Immunoprecipitation, SDS Page, Stripping Membranes

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Representative image of Lm GFP-EGD infected wildtype mCherry-Arp3 expressing usp18 C61A/C61A actr3 −/− MEFs at 12 HPI. Insets demonstrate bacteria with actin tails from two connected daughter cells. Actin (phalloidin) in blue, bacteria (green), mCherry-Arp3 (magenta) and nuclei (yellow). b Representative image of Lm GFP-EGD infected usp18 C61A/C61A actr3 −/− MEFs expressing mCherry-Arp3 3KR at 12 HPI. Insets demonstrate bacteria no longer associate with actin tails cells on both poles of connected daughter cells. Actin (phalloidin) in blue, bacteria (green), mCherry-Arp3 (magenta) and nuclei (yellow). Scale bars in image and insets are respectively 10 μm and 1 μm.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Quantification of average sensitivity to 0.05% trypsin in wildtype, Usp18 C61A/C61A , or isg15 −/− MEFs under standard tissue culture conditions; results represent mean +/-SEM from 3 individual experiments (WT n=3, CA=3, KO=3), significance determined by Tukey’s multiple comparisons. b Area under the curve analysis of trypsin sensitivity in MEFs, compared with Brown-Forsythe ANOVA test with Dunnett’s T3 post hoc. c Representative images of actin organization and Arp3 localization in mCherry-Arp3 wildtype or Arp3 3KR expressing usp18 C61A/C61A actr3 −/− MEFs. Actin (phalloidin) in green, mCherry-Arp3 in magenta, endogenous Arp3 (a-Arp3) in blue, and DAPI in yellow. Scale bars, 10 μm. d Representative image of migration pattern of Type I IFN treated (1000 U/mL for 24H) mCherry-Arp3 wildtype or Arp3 3KR expressing usp18 C61A/C61A actr3 −/− MEFs on fibronectin; scale bars, 10 μm, see SI movies 17-19. e Quantification of average population of cells present with rapidly turning over filopodia-like structure (“jazz hands”). Mean -/+ SEM from 2 individual experiments, 49-95 cells were counted and tracked per genotype per repeat. f Area under the curve analysis of MEFs with “jazz hands”, average -/+ SEM compared with twotailed unpaired t test. g Representative histological images of neonatal skin (Postnatal Day 1), H and E stain, from wildtype, usp18 C61A/C61A , and isg15 −/− animals. Scale bar = 88 μm. h Quantification of keratin layer average thickness from six neonates from each genotype; oneway ANOVA with Tukey’s post hoc test. i Quantification of average total thickness from six neonates from each genotype; one-way ANOVA with Tukey’s post-hoc test. In a , b , and f asterisks indicate p values with *p < 0.05, and ****p < 0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Expressing, Migration, Staining

Journal: PLoS ONE

Article Title: APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

doi: 10.1371/journal.pone.0120089

Figure Lengend Snippet: (A) Six selected genes were screened by specific siRNAs, and the resultant A% values at c.1303 were calculated from the WT1 cDNA clones sequenced. A3A-si1 resulted in nearly complete reversal of c.1303G>A change, with no evidence of G-to-A alterations elsewhere. Values indicate the mean +/- SEM. (B) Silencing efficiencies of different A3A -siRNAs were assessed by Taqman gene expression assay in CBMCs. (C) Phenotypic analysis of corresponding A3A- silenced CBMCs using Sanger sequencing of the WT1 -cDNA shows a virtually complete reversal of c.1303G>A change by A3A-si1 and A3A-si2, and a partial reversal by A3A-si3. (D) Quantification of the results in (C) above, as determined by Sanger sequencing of the WT1 -cDNA clones, confirming qualitative data. *** P < 0.001.

Article Snippet: Green-fluorescent protein (GFP)-tagged A3A overexpression (RG220995) and empty pCMV6-AC-GFP (PS100010) vectors were obtained from OriGene.

Techniques: Clone Assay, Gene Expression, Sequencing

Journal: PLoS ONE

Article Title: APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

doi: 10.1371/journal.pone.0120089

Figure Lengend Snippet: (A) A3A/ 10 6 GAPDH mRNA as assessed by Taqman gene expression assay in Fujioka/P31 cells 48 hours after transfection by A3A-GFP plasmid, as compared to the control vector. (B) Western Blot analysis of Fujioka cells transfected with control or A3A-GFP vector, showing exogenous GFP-tagged A3A, in addition to endogenous A3A. (C) Phenotypic analysis shows a modest effect of A3A overexpression in the induction of c.1303G>A change (i.e. increasing A% at c.1303) in Fujioka cells, as determined by Sanger sequencing of the WT1 -cDNA clones. Values indicate the mean +/- SEM. * P < 0.05.

Article Snippet: Green-fluorescent protein (GFP)-tagged A3A overexpression (RG220995) and empty pCMV6-AC-GFP (PS100010) vectors were obtained from OriGene.

Techniques: Gene Expression, Transfection, Plasmid Preparation, Control, Western Blot, Over Expression, Sequencing, Clone Assay

Journal: PLoS ONE

Article Title: APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

doi: 10.1371/journal.pone.0120089

Figure Lengend Snippet: (A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and A3A mRNA expression was assessed and normalized to GAPDH mRNA level. A3A expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.

Article Snippet: Green-fluorescent protein (GFP)-tagged A3A overexpression (RG220995) and empty pCMV6-AC-GFP (PS100010) vectors were obtained from OriGene.

Techniques: Expressing, Quantitation Assay, Western Blot

Journal: Nature communications

Article Title: APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

doi: 10.1038/ncomms7881

Figure Lengend Snippet: Figure 4 | Association of APOBEC3A gene expression with SDHB c.136C4U RNA editing in tumour samples of TCGA (a) C4U RNA editing was estimated from RNA-sequencing data for primary head and neck squamous cell carcinoma (HNSC, n ¼ 298), lung adenocarcinoma (LUAD, n ¼ 220) and secondary skin cutaneous melanoma (SKCM, n ¼ 187) tumours. Editing levels at all 213 C-bearing positions along SDHB ORF are plotted for every tumour. Mean levels at the positions (black), the c.136C site (red) and known C/T single-nucleotide polymorphism sites (green) are indicated. Inset shows SDHB c.136C4U editing levels and their mean and s.d. for tumours identified as positive for the editing. (b) Tukey’s plots of expression of some APOBEC3 (A3) and hypoxia- (LDHA and PGK1) and macrophage-associated (CD14 and MRC1) genes among SDHB c.136C4U editing-positive and -negative tumours. Error bars denote 25th percentile 1.5 interquartile range (IQR) and 75th percentile þ 1.5 IQR values. Group sizes are noted in the legend. *FDR o0.05 (edgeR exact test for differential expression).

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for cytomegalovirus (CMV) promoter-driven expression of human APOBEC3A, APOBEC3G, CDA and SDHB cDNAs, with sequences matching NCBI RefSeq sequences NM_145699.2, NM_021822.1, NM_001785.1 and NM_003000.2, respectively, for the generation of C-terminal Myc-DDK-tagged APOBEC3A and untagged APOBEC3G, CDA and SDHB proteins were obtained from OriGene (Rockville, MD; product numbers RC220995, SC122916, SC119015 and SC319204, respectively).

Techniques: Gene Expression, RNA Sequencing, Expressing, Quantitative Proteomics

Journal: Nature communications

Article Title: APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

doi: 10.1038/ncomms7881

Figure Lengend Snippet: Figure 5 | APOBEC3A induces C4U RNA editing in 293T transfectants. (a) Immunoblots showing APOBEC3A (A3A), APOBEC3G (A3G) and CDA proteins in whole-cell lysates (20 mg protein) of 293T cells transiently transfected with an empty vector (Ctrl., control) or DNA constructs for expression of A3A, A3G or CDA proteins. (b) SDHB c.136C4U RNA editing in the 293T transfectants, which were optionally treated with hypoxia and/or 600 U ml 1 type I IFN (IFN1). Mean and range for n ¼ 3 are shown. (c) Estimation of site-specific C4U RNA editing by Sanger sequencing of RT–PCR products for 30 genes in the transfectants (n ¼ 1). The detection limit for editing (5% level) is indicated. Samples without detectable editing were assigned a value of 3.8%. Chromatograms for 19 genes are shown in Supplementary Fig. 5. Chromatograms of good quality could not be obtained for C1QA and TMEM179B for the A3G and CDA transfectants, and for the GPR160 site for the normoxic A3A transfectant. (d) Chromatograms of genomic DNA (gDNA) and cDNA PCR products of normoxic A3A transfectants, indicating C4U RNA editing without C4T genomic change at positions marked with * for ASCC2, SDHB and TMEM109. Immunoblots showing ASCC2, SDHB and TMEM109 proteins in whole-cell lysates (20 mg protein) of control or A3A transfectants on the right indicate reduced protein expression in association with A3A-induced stop codons in RNA. Only a single band of signal, which corresponded to a protein of full length, was seen in all three immunoblots.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for cytomegalovirus (CMV) promoter-driven expression of human APOBEC3A, APOBEC3G, CDA and SDHB cDNAs, with sequences matching NCBI RefSeq sequences NM_145699.2, NM_021822.1, NM_001785.1 and NM_003000.2, respectively, for the generation of C-terminal Myc-DDK-tagged APOBEC3A and untagged APOBEC3G, CDA and SDHB proteins were obtained from OriGene (Rockville, MD; product numbers RC220995, SC122916, SC119015 and SC319204, respectively).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Construct, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature communications

Article Title: APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

doi: 10.1038/ncomms7881

Figure Lengend Snippet: Figure 6 | Knockdown of APOBEC3A (A3A) reduces C4U RNA editing in M1 macrophages (a) A3A and APOBEC3G (A3G) gene expression in M1 macrophages that were transfected with a nonspecific (Ctrl.) or either one (1, 2) or equimolar mix (1 þ 2) of two A3A-specific siRNAs at 100 nM concentration. Gene expression measurements are normalized to that for ACTB. (b) Immunoblot for A3A protein (23 kDa) of whole-cell lysates (10 mg protein) of two of each set of three replicate transfectants. Nonspecific signals are indicated by an asterisk (*). The signal for calnexin, a house-keeping protein, indicates total protein. (c) SDHB c.136C4U RNA-editing levels in the siRNA transfectants, which are determined by RT–qPCR. (d) Sanger sequence chromatogram traces of amplified cDNA fragments, indicating reduced site-specific RNA editing for five other genes in A3A-specific siRNA 1 compared with Ctrl. transfectants. Mean and range (n ¼ 3) are shown for a and c.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for cytomegalovirus (CMV) promoter-driven expression of human APOBEC3A, APOBEC3G, CDA and SDHB cDNAs, with sequences matching NCBI RefSeq sequences NM_145699.2, NM_021822.1, NM_001785.1 and NM_003000.2, respectively, for the generation of C-terminal Myc-DDK-tagged APOBEC3A and untagged APOBEC3G, CDA and SDHB proteins were obtained from OriGene (Rockville, MD; product numbers RC220995, SC122916, SC119015 and SC319204, respectively).

Techniques: Knockdown, Gene Expression, Transfection, Concentration Assay, Western Blot, Quantitative RT-PCR, Sequencing

Journal: Nature communications

Article Title: APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

doi: 10.1038/ncomms7881

Figure Lengend Snippet: Figure 7 | Activity of APOBEC3A (A3A) mutants in 293T transfectants (a) A3A protein level in whole-cell lysates (20 mg protein) of cells transfected with an empty vector (Ctrl.) or expression constructs for wild-type (WT) A3A or its C101S, E72D or P134A variants. (b) Cytidine deamination activity of the transfectant lysates was examined in an in vitro reaction with a 50 fluorescent dye-labelled ssDNA substrate of 40 bases (b). C4U deamination of the single cytidine residue of the substrate at position 23 followed by deglycosylation of the uridine and subsequent cleavage of the product at the abasic site was evaluated by electrophoresis of reactions of one hour duration on a polyacrylamide gel, whose fluorographic image is shown. (c) SDHB c.136C4U RNA editing in the transfectants. (d) Retrotransposition of a human LINE-1 element in a separate set of 293T transfectants. Retrotransposition, relative to the Ctrl. transfectant, was assessed with a luciferase reporter-based assay and is quantified as the ratio of firefly and Renilla luciferase activities. Mean and range (n ¼ 3) are shown for c and d.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for cytomegalovirus (CMV) promoter-driven expression of human APOBEC3A, APOBEC3G, CDA and SDHB cDNAs, with sequences matching NCBI RefSeq sequences NM_145699.2, NM_021822.1, NM_001785.1 and NM_003000.2, respectively, for the generation of C-terminal Myc-DDK-tagged APOBEC3A and untagged APOBEC3G, CDA and SDHB proteins were obtained from OriGene (Rockville, MD; product numbers RC220995, SC122916, SC119015 and SC319204, respectively).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Construct, In Vitro, Residue, Electrophoresis, Luciferase, Reporter Assay

Journal: Nature communications

Article Title: APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

doi: 10.1038/ncomms7881

Figure Lengend Snippet: Figure 8 | In vitro cytidine deamination of SDHB RNA and ssDNA by APOBEC3A (a) c.136C4U editing of an B1.1 kb exogenous SDHB ORF RNA by whole-cell lysates of control or APOBEC3A 293T transfectants. Duration of the deamination reactions and amount of lysate protein in them are noted. For some reactions, lysates were pre-heated at 85 C for 15 min. (b) c.136C4U editing of the RNA by 10 mM purified C-His6-tagged APOBEC3A protein. The reactions had 180 amole SDHB RNA and 100 nM ZnCl2. (c) Sanger sequence chromatogram traces of PCR amplified products of SDHB deamination reactions that had either 180 amole of B1.1 kb SDHB RNA or 100 amole of SDHB ssDNA of 120 b as substrate. APOBEC3A protein was present in the þ reactions at 5 and 20 mM in the reaction with RNA and DNA substrate, respectively. Reactions for b and c conducted for two hours at 37 C. Mean and range (n ¼ 3) are shown in a and b, respectively.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for cytomegalovirus (CMV) promoter-driven expression of human APOBEC3A, APOBEC3G, CDA and SDHB cDNAs, with sequences matching NCBI RefSeq sequences NM_145699.2, NM_021822.1, NM_001785.1 and NM_003000.2, respectively, for the generation of C-terminal Myc-DDK-tagged APOBEC3A and untagged APOBEC3G, CDA and SDHB proteins were obtained from OriGene (Rockville, MD; product numbers RC220995, SC122916, SC119015 and SC319204, respectively).

Techniques: In Vitro, Control, Sequencing

Journal: RNA biology

Article Title: Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes.

doi: 10.1080/15476286.2016.1184387

Figure Lengend Snippet: Figure 1. Overexpression of A3A in 293T cells induces c.136 C >U SDHB RNA editing proportional to the increased amounts of A3A plasmid. (A) SDHB c.136 C > U editing levels in 293T cells are shown. The cells are transfected with control empty vector (500 ng) or pA3A at amounts indicated in a 12-well tissue culture plate. Mean and its standard error for n D 3 are shown. (B) Immunoblot showing A3A levels in whole-cell lysates (20 mg protein) of 293T cells transiently transfected with an empty vector control (EV) or increasing amounts of plasmid DNA construct for expression of A3A (pA3A). The A3A protein expression was detected by anti-DDK tag antibody. b-Actin is used as a loading control.

Article Snippet: Gene expression construct and transfection of plasmid DNA Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3A with sequences matching NCBI RefSeq sequences NM_145699.2 and containing C-terminus MycDDK tag were obtained from OriGene (Rockville, MD, product number RC220995).

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Western Blot, Construct, Expressing

Journal: RNA biology

Article Title: Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes.

doi: 10.1080/15476286.2016.1184387

Figure Lengend Snippet: Figure 2. Sanger validation of C > U RNA editing sites in primary cells identified by low-stringency RNA seq analysis of 293T/A3A cells. RNA editing at the highlighted Cs was validated in monocyte/enriched PBMCs (MEPs; n D 3 donors) treated with hypoxia and IFN1 (HI) for 24 h, in 7 of 19 tested genes. C > T(U) editing is characterized by emergence of a secondary T peak (red) accompanied by a reduction in height of C peak (blue). MEP-N shows lack of editing in a representative sequence chromato- gram from control MEP cells maintained in normoxia without IFN1 treatment. Chromatograms for the remaining 12 RNA editing sites that could not be validated in MEPs are shown Fig. S2.

Article Snippet: Gene expression construct and transfection of plasmid DNA Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3A with sequences matching NCBI RefSeq sequences NM_145699.2 and containing C-terminus MycDDK tag were obtained from OriGene (Rockville, MD, product number RC220995).

Techniques: Biomarker Discovery, RNA Sequencing, Sequencing, Control

Journal: RNA biology

Article Title: Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes.

doi: 10.1080/15476286.2016.1184387

Figure Lengend Snippet: Figure 3. Salient characteristics of C > (U)RNA editing by A3A in 293T cells (A) Mean and range of editing level at the 4,373 sites identified as targets for A3A-mediated editing are shown for the 3 A3A 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 lengths in bases of inverted repeat sequences flanking the editing sites.

Article Snippet: Gene expression construct and transfection of plasmid DNA Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3A with sequences matching NCBI RefSeq sequences NM_145699.2 and containing C-terminus MycDDK tag were obtained from OriGene (Rockville, MD, product number RC220995).

Techniques: Transfection, Sequencing