Journal: Nucleic acids research

Article Title: Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome.

doi: 10.1093/nar/gkac1238

Figure Lengend Snippet: Figure 3. A3A mRNA expression in human airway and lung cells. (A) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF- under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy num- bers of A3A relative to those of the housekeeping gene RPP40 are shown (n = 3). (B) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown (n = 3). The error bars indicate the + SD values.

Article Snippet: Human nasal epithelial primary cells (HNEpCs) and human type II alveolar epithelial (AT2) cells were purchased from PromoCell and Accegen Biotechnology, respectively.

Techniques: Expressing, Control, Cell Culture

Journal: bioRxiv

Article Title: Epigenetic deregulation of IFN and WNT pathways in AT2 cells impairs alveolar regeneration (in COPD)

doi: 10.1101/2023.10.22.563483

Figure Lengend Snippet: A . LINE methylation levels in A549 cells treated with the indicated amounts of 5-Aza-2’- deoxycytidine (AZA) and measured at three CpG sites by Mass Array. B-D . Fold-change in gene expression of selected genes in AT2 cells in COPD (RNA-seq) and in A549 cells treated with 0.5µM AZA (RT-qPCR) compared to control samples. Genes were selected based on the presence of a DMR and deregulation in AT2 cells in COPD. Left, RNA-seq data from AT2 cells (no COPD, blue, n=3; COPD II-IV, green, n=5), right, A549 treated with AZA (orange, n=3) compared to control DMSO treated cells (grey, n=3). The group median is shown as a black bar. Selected genes from the WNT/β-catenin pathway ( B ), antimicrobial peptides, alveolar progenitor ( C ) and airway epithelial markers ( D ) are shown. RNA-seq FDRs were calculated using DESeq2 with negative binomial generalized linear model (GLM) and Wald statistics. *: FDR <0.05; **: FDR <0.01. Gene expression was measured by RT-qPCR using DMSO treatment as control (grey) and RPLP0 as housekeeping gene. For A549 samples, each point represents the mean of two technical replicates, and bars represent the median of 3 independent experiments (n=3). Statistical analysis was performed by paired t-test, FDR-corrected using the Benjamini, Krieger, and Yekutieli two-stage set-up method. *p-value < 0.05; **p-value < 0.01. E . Graphical representation of the IRF9 locus. In dark blue is the IRF9 coding region, with introns and exons represented by dashed and solid lines, respectively. At the bottom, arrows represent individual gRNAs targeting the IRF9 promoter (orange) and gene body (magenta) regions, with overlapping lines representing the groups of gRNAs that comprise mix 1 and mix 2, respectively. Bisulfite-PCR amplicons targeting IRF9 (blue bars) are depicted as blue boxes, with P1-4 targeting the IRF9 promoter region, and GB1-2 targeting the gene body region. Individual CpG sites are depicted by black bars below the IRF9 coding region, with the light and dark green overlapping region displaying the core and extended differentially methylated regions, respectively, identified in the AT2 COPD TWGBS data. Genomic positions were extracted from human genome assembly 38 (hg38) using the UCSC genome. F . CpG-methylation percentage at individual IRF9 CpG sites in epi-edited A549 cells. The percentage CpG-methylation for mix 1, 2, and 1+2 (orange, magenta, and dark grey) transfected samples are plotted separately against the pUC19 mock (mock) and non-targeted (NTC) transfection controls. For each sample, the opaque line plots the mean value, and the faint surrounding area plots the observed range across repeats. The bisulfite PCR targets are displayed across the top, showing which CpG-sites are sequenced by each target, and how these sites relate to the genomic locations of the sequencing targets seen in panel.

Article Snippet: The human AT2-like cell line A549 (CCL-185, ATCC) was grown in Ham’s F12 medium (PAN Biotech, P04-14550) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270106), 1% Glutamax (Gibco, 35050061) and 1% penicillin-streptomycin (Fisher Scientific, 15140122) at 37C in 5% CO 2 atmosphere, as recommended to preserve the AT2-like phenotype ( ).

Techniques: Methylation, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Control, CpG Methylation Assay, Transfection, Sequencing

Journal: bioRxiv

Article Title: Epigenetic deregulation of IFN and WNT pathways in AT2 cells impairs alveolar regeneration (in COPD)

doi: 10.1101/2023.10.22.563483

Figure Lengend Snippet: A . Relative changes in expression (2 -ΔΔC T ) of the indicated IFN pathway genes in A549 cells upon 5-Aza-2’-deoxycytidine (AZA, orange) treatment. Gene expression was measured by RT-qPCR using DMSO treatment as control (grey) and RPLP0 as housekeeping gene. Each point represents the mean of two technical replicates, and bars represent the median of 3 independent experiments (n=3). Paired t-test, FDR corrected using two-stage set-up method of Benjamini, Krieger, and Yekutieli. *p-value < 0.05; **p-value < 0.01. B. Cytoscape analysis of components of the IFN pathway identifying IRF9 as a master regulator. Nodes are connected based on their protein-protein interaction annotation in the categories binding (blue line) or activation (grey arrow) in STRING data base. The node size represents the betweenness centrality within the network. DEG are shown as triangles and log2(fold-change) in severe COPD is indicated by the node color. Red labeled nodes exhibit a negative Spearmann correlation (< −0.5) with promoter proximal associated DMRs. C . Scatter plots showing correlation between gene expression and methylation of promoter proximal DMRs for IRF9. Each dot represents an individual donor. Dots are color coded according to the disease state. Gene expression is illustrated as normalized counts. Methylation is illustrated as the average beta value of the corresponding DMR. D . Detailed view of the IRF9 locus, featuring a core and extended DMR (light and dark green boxes) identified between no COPD and severe COPD. Orange boxes represent the location of individual gRNAs used for targeting the promoter (P, mix 1), and dark purple boxes indicate the location of individual gRNAs targeting the gene body region (GB, mix 2). E-G Targeted DNA demethylation of IRF9 using CRISPR-based epigenetic editing in A549 cells. E. Box plots displaying percentage DNA methylation at CpG sites across the IRF9 promoter (left) or gene body regions (right) after transfection with the dCas9-VPR-mTet3 demethylating construct and IRF9 targeting gRNA mixes (or non-targeted control; NTC, which contained dCas9-VPR-mTet3 but no gRNA). For each sample, average methylation was calculated per CpG from 3 independent biological replicates and was aggregated into bins containing CpGs from either the promoter or the gene body target regions. Boxes represent the median and IQR of the data, and whiskers represent the full range of non-outlier values. Statistical significance of data was analyzed using the Kruskal-Wallis multiple comparison test, followed by Dunn’s post-hoc analysis comparing each sample against the NTC (total 3 comparisons), and adjusting p-values using the Benjamini-Hochberg correction method (*p-value<0.05; **p-value<0.01; ***p-value<0.001; ****p-value<0.0001). F-G. Relative changes in expression (2 -ΔΔC T ) of IRF9 ( F ) and a panel of its downstream targets (G) . Displayed are the mean fold-changes in gene expression induced by transfection with the dCas9-VPR-mTet3 demethylating construct and IRF9 targeting gRNA mixes (or non-targeted control; NTC) normalized to mock transfection control. Biological replicates data (n=3) is represented by individual data points, and standard deviation by the error bars. Statistical significance of data was analyzed by Kruskal-Wallis multiple comparison tests, followed by Dunns post-hoc analysis test with each sample against the NTC (total 3 comparisons), and adjusting p-values using the Benjamini-Hochberg correction method (p-value<0.05; **p-value<0.01; ***p-value<0.001; ****p-value<0.0001).

Article Snippet: The human AT2-like cell line A549 (CCL-185, ATCC) was grown in Ham’s F12 medium (PAN Biotech, P04-14550) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270106), 1% Glutamax (Gibco, 35050061) and 1% penicillin-streptomycin (Fisher Scientific, 15140122) at 37C in 5% CO 2 atmosphere, as recommended to preserve the AT2-like phenotype ( ).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Binding Assay, Activation Assay, Labeling, Methylation, CRISPR, DNA Methylation Assay, Transfection, Construct, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome

doi: 10.1093/nar/gkac1238

Figure Lengend Snippet: A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.

Article Snippet: Human nasal epithelial primary cells (HNEpCs) and human type II alveolar epithelial (AT2) cells were purchased from PromoCell and Accegen Biotechnology, respectively.

Techniques: Expressing, Control, Cell Culture