Journal: bioRxiv

Article Title: Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers

doi: 10.1101/2022.10.05.510981

Figure Lengend Snippet: A . Triplicate HOTThet reporter mice were instilled with PM 10 from Marylebone St. and LacZ staining was performed in lungs (upper panels). Cryosections derived from the same samples were subjected to F4/80 immunohistochemistry (lower panels). B . Effect of 10 μg/mL Marylebone Road PM10 (MR-PM10) on PAFR expression in human primary nasal epithelial cells. Data are from 5 separate experiments and summarised as median fluorescent intensity (MFI). Figure represents the median value and p values are calculated by Mann Whitney test. C . Effect of 10 μg/mL Marylebone Road PM10 (MR-PM 10 ) on pneumococcal adhesion to human primary nasal epithelial cells (HPNEpC) and PAFR blocker CV3988 on MR-PM 10 stimulated pneumococcal adhesion to HPNEpC. Data are from 6 separate experiments. Data are summarised as median IQR Kruskal– Wallis with post hoc multiple comparison testing. D . Effect of anti-oxidant N-acetylcysteine on MR-PM10 stimulated pneumococcal adhesion to HPNEpC. Data are from 7 separate experiments and are summarised as median IQR Kruskal–Wallis with post hoc multiple comparison testing. Black arrow indicates reporter activation.

Article Snippet: Human primary nasal epithelial cells (HPNEpC) were purchased from PromoCell® (Heidelberg, Germany), and maintained in airway epithelial cell growth medium, with supplement kit, Primocin (InvivoGen, San Diego, USA), and 10% FBS.

Techniques: Staining, Derivative Assay, Immunohistochemistry, Expressing, MANN-WHITNEY, Activation Assay

Journal: PLoS ONE

Article Title: Susceptibility of Human Head and Neck Cancer Cells to Combined Inhibition of Glutathione and Thioredoxin Metabolism

doi: 10.1371/journal.pone.0048175

Figure Lengend Snippet: A: Exponential growing and confluent FaDu, Cal-27 and SCC-25 cells were treated with 0.5 µM AUR and/or 1 mM BSO for 24 h and analyzed for clonogenic survival. Clonogenic cell survival data were normalized to exponentially growing and confluent control cells (not shown). B: FaDu and HNEpC cells were treated with BSO+AUR and the number of viable attached cells was counted after 24 h. Numbers of viable BSO+AUR-treated cells were normalized to their respective controls (CON). Error bars represent the standard error of the mean (SEM) of N = 3 experiments. *, p<0.05 versus EXP; ¥, p<0.05 versus CON.

Article Snippet: HNEpC cells were obtained from PromoCell (Heidelberg, Germany).

Techniques:

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Flow cytometry analysis of healthy human BM uncovers a heterogeneous Lin−CD66b+CD117+ hNeP fraction. Dump antibody cocktail contains antibodies against markers that are expressed by: HSC (CD90 (Thy1)), lymphocytes and their progenitors (CD3, CD19, CD56, CD161, CD7, CD127 (IL-7Rα)), Erythrocytes and their progenitors (CD41, CD235a (Glycophorin A)), Eosinophils/Basophils and their progenitors (Siglec 8, FcεRIα, , CD125 (IL-5Rα)), CMP/GMP and monocyte progenitors (CD123 (IL-3Rα)), DCs and macrophages (CD11c, CD169). (B) ScRNA-Seq analysis of Lin−CD66b+CD117+ hNeP cells reveals two major subpopulations, Subset A and Subset B. 20,000 hNeP cells were FACS sorted from healthy human BM for scRNA-Seq. Heatmap shows top 40 differentially expressed genes in each cluster. Log2 Fold Change of each gene expression is relative to the entire dataset. 2 biological triplicates, 2 technical replicates. (C) Lin−CD66b+CD117+ hNeP were divided into CD34+ subset and CD34−subset by flow cytometry. Confocal microscopy was used to detect Ki67 localization (red) within the nuclei (blue) in CD34+ subset and CD34−subset using antibodies to Ki67 and Hoechst. IgG stained cells served as negative control. Bar: 5µm.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Gene Expression, Confocal Microscopy, Staining, Negative Control

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Scheme showing the experimental procedure. CD34+ hNeP and CD34−hNeP subsets identified in Figure 5C were sorted from healthy human BM, and adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human hNeP progenitor cells. After the transfer, peripheral blood was collected from each recipient via saphenous vein for flow cytometry on Day (D) 5, 7, 14, 28 (D5, D7, D14, D28), respectively. (B) Representative plots show the appearance of monocytes (CD86+ CD66b−), neutrophils (CD86−Siglec 8−CD66b+), eosinophils (Siglec 8+), and lymphocytes (hLy+) in each recipient group at the time points indicated. hLy antibody cocktail contains CD3, CD19, and CD56. N = 10 mice for each group.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) hNeP is increased in melanoma patient blood. hNeP frequency was detected by flow cytometry in peripheral blood collected from healthy donors (N = 5) and melanoma patients (N = 5). Error bars indicate mean ± SEM. (B) Left, scheme showing the experiment procedure. CD34+ hNeP subset, CD34−hNeP subset, and human cMoP were FACS sorted from healthy human BM. The 3 populations were adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human progenitor cells. Blank control group received only PBS for adoptive transfer. The next day, 1 × 106 143B human osteosarcoma cells were SubQ injected into each recipient mouse. Right, the tumor volume in each recipient was measured at 10d post-injection. N = 5 mice in each group. Error bars indicate mean ± SEM. (C) CD34+ hNeP cells blunt T cell activation. FACS sorting strategies for CD34+ hNeP and CD34−hNeP are shown in Figure 5A and ​andCC using using flow cytometry; FACS sorting strategies for mature neutrophils is shown in Figure S9A using flow cytometry. Histogram overlay of CD69 for CD3+ T cells cultured with CD34+ hNeP, CD34−hNeP, and mature neutrophils, in the presence of anti-CD3 for 24 hours. CD3+ T cells alone cultured without anti-CD3 served as the negative control group, and CD3+ T cells alone cultured with anti-CD3, CD28 served as the positive control group for T cell activation.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Control, Adoptive Transfer Assay, Injection, Activation Assay, Cell Culture, Negative Control, Positive Control

Journal: The World Allergy Organization Journal

Article Title: MicroRNA-375-mediated regulation of ILC2 cells through TSLP in allergic rhinitis

doi: 10.1016/j.waojou.2020.100451

Figure Lengend Snippet: The expression of thymic stromal lymphopoietin (TSLP) by human nasal epithelial cell (HNECs) after transfected with miR-375 mimics, miR-control, miR-375 inhibitor or AG490 (JAK2-STAT3 inhibitor) in Figure A. The effect of Th2 cytokines on the expression of TSLP by HNECs was shown in Figure B. HDM, Dermatophagoides farinae

Article Snippet: Human nasal epithelial cell line (HNECs) was obtained (ATCC, Rockville, MD, USA) and cultured in bronchial epithelial cell basal medium (BEBM, Lonza, Walkersville, MD, USA) at 37 °C in a 5% CO 2 -humidified chamber.

Techniques: Expressing, Transfection, Control

Journal: The World Allergy Organization Journal

Article Title: MicroRNA-375-mediated regulation of ILC2 cells through TSLP in allergic rhinitis

doi: 10.1016/j.waojou.2020.100451

Figure Lengend Snippet: The expression of type II cytokines by type II innate lymphoid cells (ILC2) in the coculture system with human nasal epithelial cell (HNECs) in different combination of stimulators. AG490, JAK inhibitor. TSLP, thymic stromal lymphopoietin

Article Snippet: Human nasal epithelial cell line (HNECs) was obtained (ATCC, Rockville, MD, USA) and cultured in bronchial epithelial cell basal medium (BEBM, Lonza, Walkersville, MD, USA) at 37 °C in a 5% CO 2 -humidified chamber.

Techniques: Expressing

Journal: Cell reports

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1016/j.celrep.2023.113478

Figure Lengend Snippet: (A) Representative brightfield images of A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Scale bars, 200 μm. (B) Top, western blotting analysis of whole cell lysates of A549ACE2 or A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Relative ratios of TP53/ACTB (normalized to EGFP -Dox) are annotated below TP53. Bottom, western blotting analysis of supernatants (SUP) of the corresponding cell constructs and treatments. (C) MSigDB pathway enrichment analysis of differential RNA-seq profiles upon doxycycline-induced expression (48 h, 1 μg/mL) of SARS-CoV-2 ancestral or Delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53ko pInducer20 cells. n = 2 technical replicates/group. (D) Cell viability as assessed by CellTiter-Glo in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 72 h (mean ± SEM). The Welch two-sample t test with Holm p value correction was used to assess statistical significance: ***p < 0.001; **p < 0.01. n = 5 technical replicates/group. (E) RNA levels of CDKN1A by RT-qPCR in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 48 h (mean ± SEM). The Welch two-sample t test with Benjamini-Hochberg p value correction was used to assess statistical significance relative to the EGFP -Dox condition: ***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant. n = 4 technical replicates/group. (F) Senescence-associated beta-galactosidase activity in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 48 h (mean ± SEM). The Welch two-sample t test with Holm p value correction was used to assess statistical significance: **p < 0.01; n.s., not significant. n = 14 technical replicates/group. (G) Left, ATAC-seq volcano plots of ancestral or Delta spike vs. EGFP expression in A549ACE2 pInducer20 cells 48 h after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) ATAC-seq peaks upon doxycycline-induced expression (48 h, 1 μg/mL) of SARS-CoV-2 ancestral or Delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53ko pInducer20 cells. Black triangles, peaks with increasing accessibility. Red circles, peaks with decreasing accessibility. n = 2 technical replicates/group. (H) Left, heatmap of relative levels of differentially expressed microRNAs (FDR < 0.05 and base mean > 10) from ancestral or Delta spike vs. EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells 48 h after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) differentially expressed microRNAs upon doxycycline-induced expression (48 h, 1 μg/mL) of ancestral or Delta spike vs. EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells. Black triangles, significantly upregulated microRNAs. Red circles, significantly downregulated microRNAs. n = 2 technical replicates/group. (I) Heatmap of relative levels of cytokines in supernatants of A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with 1 μg/mL doxycycline for 48 h. The Welch two-sample t test with Benjamini-Hochberg p value correction was used to assess statistical significance. Sig, FDR < 0.05. n = 4 technical replicates/group. (J) Representative brightfield images of HNEpC (human nasal epithelial cell)-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Scale bars, 200 μm. (K) Western blotting analysis of supernatants of HNEpC-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h.

Article Snippet: A549, A549 TP53ko, and HNEpC cells were transduced with pLEX307-ACE2-blast (Addgene #158449) to generate A549ACE2, A549ACE2 TP53ko, and HNEpC-ACE2 cell lines.

Techniques: Transduction, Construct, Western Blot, RNA Sequencing, Expressing, Quantitative RT-PCR, Activity Assay

Journal: Cell reports

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1016/j.celrep.2023.113478

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A549, A549 TP53ko, and HNEpC cells were transduced with pLEX307-ACE2-blast (Addgene #158449) to generate A549ACE2, A549ACE2 TP53ko, and HNEpC-ACE2 cell lines.

Techniques: FLAG-tag, Luciferase, Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Activity Assay, Software, CRISPR