primary normal human lung fibroblasts nhlf  (ATCC)

Journal: Bioengineering

Article Title: A 3D Epithelial–Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix

doi: 10.3390/bioengineering11090946

Figure Lengend Snippet: Schematic representation of the model assembly steps. Workflow for the preparation and analysis of the 3D co-culture of primary human fibroblasts and epithelial cells with lung ECM hydrogels. Created with biorender.com.

Article Snippet: Primary human lung fibroblasts ( n = 5) were cultured in fibroblast medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% GlutaMAX (Gibco), and 0.17 mM ascorbic acid.

Techniques: Co-Culture Assay

Journal: Bioengineering

Article Title: A 3D Epithelial–Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix

doi: 10.3390/bioengineering11090946

Figure Lengend Snippet: Collagen IV coating of collagen I and porcine lung ECM hydrogels on cell viability. ( A ) Collagen I-embedded fibroblast viability 1 day after collagen IV staining procedure. Calcein AM (green) staining of live fibroblasts; propidium iodine (PI; red) staining of dead cells; DAPI (blue) indicating nuclei; and a brightfield overview. The scale bars represent 200 μm. ( B ) Immunohistochemical staining of collagen IV on top of fibroblast-seeded collagen I and 10 mg/mL porcine lung ECM hydrogels 24 h after coating. The scale bars represent 100 μm. Results are representative for all experiments ( n = 2).

Article Snippet: Primary human lung fibroblasts ( n = 5) were cultured in fibroblast medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% GlutaMAX (Gibco), and 0.17 mM ascorbic acid.

Techniques: Staining, Immunohistochemical staining

Journal: Bioengineering

Article Title: A 3D Epithelial–Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix

doi: 10.3390/bioengineering11090946

Figure Lengend Snippet: Model assembly with porcine lung ECM hydrogel and its contraction over time. ( A ) Macroscopic view of the 10 and 20 mg/mL porcine lung ECM hydrogel 3D co-culture model: side view directly after full assembly; top view after 4 days submerged culture; 7-day ALI culture; and 14-day ALI culture. ( B ) Microscopic (20×) images after initial seeding of epithelial cells on and fibroblast seeding in ECM hydrogel showing reduced visibility after hydrogel contraction. ( C ) Brightfield and fluorescent microscopic images (10×): images 1 and 5 days after epithelial cell seeding on top of fibroblast-containing or empty 10 mg/mL porcine lung ECM hydrogels. Cells are stained with DAPI to visualize nuclei. ECM: extracellular matrix, ALI: air–liquid interface, FN: fibronectin, BSA: bovine serum albumin, DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: Primary human lung fibroblasts ( n = 5) were cultured in fibroblast medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% GlutaMAX (Gibco), and 0.17 mM ascorbic acid.

Techniques: Co-Culture Assay, Staining

Journal: Bioengineering

Article Title: A 3D Epithelial–Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix

doi: 10.3390/bioengineering11090946

Figure Lengend Snippet: Histological analysis of primary airway epithelial, fibroblast, and porcine lung ECM co-culture model of cell differentiation prior to and after air–liquid interface (ALI) culture. Tubulin immunostaining (ciliated cells), Alcian blue staining (goblet cells), MUC5AC immunostaining (club cells), and KRT5 immunostaining (basal cells). Submerged culture was fixed after epithelial cells reached confluence, whereas ALI culture exposed the submerged cultured cells to air for 14 days before fixation. ( A ) Representative images of sections (40× magnification) of submerged- and ALI-cultured porcine ECM (conc. 10 mg/mL) epithelial cell fibroblast co-culture model. ( B ). Quantified differentiated epithelial cells on membranes coated with collagen I-FN-BSA. ( C ) Representative images of sections (40× magnification) of porcine ECM (conc. 20 mg/mL) epithelial cell fibroblast co-culture model. ( D ) Quantified differentiated epithelial cells on membranes coated with collagen IV. Scale bars are 50 µm. All data are shown as means with all available data plotted individually ( n = 1–5). ECM: extracellular matrix, ALI: air–liquid interface, MUC5AC: mucin-5AC, KRT5: keratin 5. Made with Biorender.com.

Article Snippet: Primary human lung fibroblasts ( n = 5) were cultured in fibroblast medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% GlutaMAX (Gibco), and 0.17 mM ascorbic acid.

Techniques: Co-Culture Assay, Cell Differentiation, Immunostaining, Staining, Cell Culture

Journal: Bioengineering

Article Title: A 3D Epithelial–Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix

doi: 10.3390/bioengineering11090946

Figure Lengend Snippet: Histological analysis of primary airway epithelial, fibroblast, and human lung ECM (HECM) co-culture model of cell differentiation prior to and after air–liquid interface (ALI) culture. Tubulin immunostaining (ciliated cells), Alcian blue staining (goblet cells), MUC5AC immunostaining (club cells), and KRT5 immunostaining (basal cells). Submerged culture was fixed after epithelial cells reached confluence, whereas ALI culture exposed the submerged-cultured cells to air for 14 days before fixation. ( A ) Representative images of sections (40× magnification) of submerged- and ALI-cultured control human lung ECM hydrogel epithelial cell fibroblast co-culture model. ( B ). Quantified differentiated epithelial cells on membranes coated with collagen I-FN-BSA. ( C ). Representative images of sections (40× magnification) of submerged- and ALI-cultured COPD human lung ECM hydrogel epithelial cell fibroblast co-culture model. ( D ) Quantified differentiated epithelial cells on membranes coated with collagen IV. Scale bars are 50 µm. All data are shown as means with all available data plotted individually ( n = 5). ALI: air–liquid interface, MUC5AC: mucin-5AC, KRT5: keratin 5. Made with Biorender.com.

Article Snippet: Primary human lung fibroblasts ( n = 5) were cultured in fibroblast medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% GlutaMAX (Gibco), and 0.17 mM ascorbic acid.

Techniques: Co-Culture Assay, Cell Differentiation, Immunostaining, Staining, Cell Culture, Control

Journal: Nucleic Acids Research

Article Title: Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler

doi: 10.1093/nar/gkae615

Figure Lengend Snippet: Upregulation of Alu expression by e1a in IMR90 cells. ( A ) Venn-diagram showing the number of expressed Alu elements in dl 1500-, dl 312- and mock-infected cells. The whole experiment was performed in duplicate. Expressed Alu s included elements whose expression was detected by the pipeline in at least one replicate. ( B ) Average Alu expression profiles in the presence/absence of e1a, generated from normalized read counts (Counts Per Million, CPM) of the 1805 expression-positive Alu s. Shown in the lower part of the panel is the structure of a typical full-length Alu element. The approximate positions of the A and B box internal control regions (yellow bars) are indicated above, those of internal and terminal poly(dA) motifs are indicated by white bars. The approximate position and extension of Alu internal sequence elements are indicated below (bp, base pairs). The upper graph reports the average read count for both replicates of each sample ( dl 1500, dl 312 and mock), labelled by different colours as indicated. The vertical dashed line marks the position of the Alu transcription start site (TSS). ( C ) Base resolution expression profiles, shown as Integrated Genome Browser views , of 4 Alu s representative of different types of response to e1a. For the Alu in the first view on the left (AluSp_chr4), no substantial expression changes were observed under the different conditions. The Alu in the second view from the left (AluSq2_chr6) is detected as expressed in dl 312- and mock-infected cells and its expression is strongly increased by e1a. The expression of the Alu in the third view from the left (AluSp_chr8) is only detected in dl 1500-infected cells. The rightmost view (AluSx1_chr9) illustrates one of the very few examples of e1a-dependent downregulation. Orange boxes represent the orientation of repetitive elements as evidenced by the RepeatMasker track. The chromosomal coordinates of each annotated Alu are shown above each view. Bigwig RNA-seq data are normalized as Counts Per Million. The profiles of both replicates on (+) and (–) strands are shown for each sample (mock-, dl 312- and dl 1500-infected cells).

Article Snippet: IMR90 primary human fetal lung fibroblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Infection, Generated, Control, Sequencing, RNA Sequencing Assay

Journal: Nucleic Acids Research

Article Title: Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler

doi: 10.1093/nar/gkae615

Figure Lengend Snippet: Dependence of Alu upregulation on e1a interaction with chromatin regulators. ( A ) Heatmap showing increased (red) or decreased (blue) expression of Alu elements triggered by wt e1a or e1a mutants defective in interaction with RB (e1a_RB-b − ), p300 (e1a_p300-b − ) or p400 (e1a_p400-b − ), as compared to mock-infected cells. Boxed above each heatmap are the numbers of differentially expressed Alu s (log 2 fold-change ≥ 0.5 or ≤ –0.5 and an adjusted P -value < 0.05). The experiment was performed in two biological replicates. ( B ) Expression levels of two individual Alu s as measured by RT-qPCR (upper graphs) and RNA-seq (lower views). Fold changes estimated by RT-qPCR are relative to the expression in mock-infected cells, after normalization to U1 snRNA gene expression. Primers were chosen to target the unique sequence of Alu elements within the 3′ trailer region. RT-qPCR data relative to each independent experiment are represented as dots. Indicated by horizontal bars are the means ± standard deviation between the replicates. RNA-seq data (lower subpanels) are presented as genome browser views of the same Alu elements analysed in the upper plots. Orange boxes represent the orientation of repetitive elements as evidenced by the RepeatMasker track. The chromosomal coordinates of each annotated Alu are shown in the upper part of each subpanel. Bigwig tracks are normalized per CPM. ( C ) Expression changes of 7SL RNA (left graph) and U6 snRNA (right graph) genes induced by either wt e1a or e1a_p400-b − mutant, as measured by RT-qPCR. Fold change is relative to mock-infected cells, after normalization to U1 snRNA gene expression. RT-qPCR data from each of two independent experiments are represented as dots. Indicated by horizontal bars are the means ± standard deviation between the replicates. ( D ) Genome browser views of the expression of RN7SL1, RPPH1, tRNA-His-GTG-1–1 (GtRNAdb), RNU6-9 and RN7SK genes, coding for 7SL RNA, Ribonuclease P RNA component H1, tRNA Gly (GGA), U6 snRNA and 7SK RNA, respectively. Expression profiles are based on RNA-seq analysis of IMR90 cells infected as indicated on the left. ( E ) Heatmap and enrichment profiles (normalized read tags) of Bdp1 ChIP-seq occupancy at differentially expressed Alu s (DE ep Alus ) and ep Alus in IMR90 infected with dl 312, dl 1500 and p400-b − viruses. ( F ) Genome browser views Bdp1 ChIP-seq data of two highly dl 1500-induced Alu elements as evidenced by the RepeatMasker track. The chromosomal coordinates of each annotated Alu are shown above each view. Bigwig tracks are normalized for the library size.

Article Snippet: IMR90 primary human fetal lung fibroblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Infection, Quantitative RT-PCR, RNA Sequencing Assay, Sequencing, Standard Deviation, Mutagenesis, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler

doi: 10.1093/nar/gkae615

Figure Lengend Snippet: Genome-wide location analysis of TFIIIC and TFIIIB in the presence/absence of e1a. ( A ) Heatmap of TFIIIC (GTF3C2) and Bdp1 spanning ±1 kb across all TFIIIC-bound sites in mock- and dl 1500-infected cells. Clusters 1 to 4 were created by combinatorial clustering of the two factors across all regions bound. Color bar scale with increasing shades of color stands for increasing enrichment (normalized read tags). ( B ) Shown on the left is the word cloud analysis of repetitive elements associated with regions occupied by TFIIIC and Bdp1 in the four clusters. Font size reflects enrichment for the indicated term. Reported on the right are the results of sitepro analysis of TFIIIC and Bdp1 enrichment (normalized read tags) for each cluster reported in panel A. Enrichment is shown spanning 2 kb from the center of the peaks. ( C ) Shown in the upper part of the panel are the average ChIP-seq enrichment profiles (normalized read tags) of the TFIIIC 110 kDa subunit (left) or the Bdp1 component of TFIIIB (right) in either mock-infected or dl 1500-infected IMR90 cells across the 1805 ep Alu s and across random Alu s. Reported below the plots are heatmaps of TFIIIC and Bdp1 enrichment at the same Alu s, sorted according to their expression level in dl 1500-infected cells (top, high expression; bottom, low expression). ( D ) Enrichment profiles (normalized read tags) of TFIIIC and Bdp1, in either mock-infected or dl 1500-infected IMR90 cells, at differentially expressed Alu s whose expression levels in the presence of e1a falls in the first quartile (Q1 DE ep Alu ), sorted according to their expression level in dl 1500-infected cells (top, high expression; bottom, low expression).

Article Snippet: IMR90 primary human fetal lung fibroblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, Infection, ChIP-sequencing, Expressing

Journal: Nucleic Acids Research

Article Title: Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler

doi: 10.1093/nar/gkae615

Figure Lengend Snippet: Histone modification and chromatin regulator enrichment profiles of ep Alu s in the presence/absence of e1a. ( A ) Shown in the upper graphs are the average ChIP-seq enrichment (–log 10 of the Poisson P -value) profiles of (from left to right) H3K18ac, H3K9ac, H3K27ac and H3K4me1 across the 1805 ep Alu s in either mock-infected or dl 1500-infected IMR90 cells. Reported below the plots are the heatmaps of the same histone modification enrichments, with ep Alus and random Alus ranked according to enrichment expressed as –log 10 of the Poisson P -value. ( B ) Shown in the upper part of the panel are the average ChIP-seq enrichment profiles (–log 10 of the Poisson P -value) of EP300 (left) and RB1 (right) in either mock-infected or dl 1500-infected IMR90 cells across the 1805 ep Alu s and across random Alu s. Reported below the plots are heatmaps of EP300 and RB1 association to the same Alu s, sorted according to their expression level in dl 1500-infected cells (top, high expression; bottom, low expression).

Article Snippet: IMR90 primary human fetal lung fibroblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Modification, ChIP-sequencing, Infection, Expressing

Journal: Nucleic Acids Research

Article Title: Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler

doi: 10.1093/nar/gkae615

Figure Lengend Snippet: Enrichment of EP400 and H2A.Z at ep Alu s and effects of EP400 depletion. ( A ) ChIP-seq enrichment profiles of Pol III (RPC155 subunit), TFIIIC and EP400 (p400) at the 285 Alu elements that are expression-positive in both IMR90 (this study) and K562 cells ( , ). Plotheatmap of ChIP-seq data. From left to right: Pol III enrichment in K562 cells, TFIIIC enrichment in K562 cells, TFIIIC enrichment in mock-infected and dl 1500-infected IMR90 cells and EP400 enrichment in K562 cells. Ranking is according to enrichment of Pol III in K562 cells reported as -log 10 of the Poisson P -value. ( B ) Plotheatmap of ChIP-seq enrichment of EP400 (left) and the H2A.Z histone variant (right) in K562 cells ( , ) across either the 1805 IMR90 ep Alu s or random Alu s. Ranking is according to enrichment of EP400 in K562 cells reported as -log 10 of the Poisson P -value. ( C ) Plotheatmap of ChIP-seq enrichment of EP400 (left) and the H2A.Z histone variant (right) in K562 cells across either the 3764 Alu s detected as expressed in K562 cells or random Alu s. Ranking is according to enrichment of p400 in K562 cells reported as -log 10 of the Poisson P -value. ( D ) Schematic representation of the protocol of siRNA-mediated EP400 knock down (KD) followed adenoviral infection (time of each incubation is reported). ( E ) RT-qPCR for measuring expression of two ep Alu loci (the same as in Figure ) comparing mock-, e1a_p400-b — and dl 1500-infection in conditions of absence of silencing RNA (non-siRNA) or presence of siRNA against p400 (siEP400) compared to a scramble set of siRNA control (siCTRL). Standard error bars are indicated, as a result of two biological replicates.

Article Snippet: IMR90 primary human fetal lung fibroblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: ChIP-sequencing, Expressing, Infection, Variant Assay, Knockdown, Incubation, Quantitative RT-PCR, Control