Journal: Nutrients

Article Title: Essential Oils, Pituranthos chloranthus and Teucrium ramosissimum , Chemosensitize Resistant Human Uterine Sarcoma MES-SA/Dx5 Cells to Doxorubicin by Inducing Apoptosis and Targeting P-Glycoprotein

doi: 10.3390/nu13051719

Figure Lengend Snippet: Effects of Pituranthos chloranthus (PC) and Teucrium ramosissimum Desf. (TR) extracts on normal primary human uterine fibroblast cells (HUF) and primary murine Bone Marrow-Derived Macrophages (BMDM) viability. After treatment of primary HUF and murine BMDM with increasing concentrations (0–100 µg/mL) of PC and TR for 72 h, the percentage of viable cells was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ( A ) Dose–response curves of PC-treated HUF (left panel) and TR-treated HUF (right panel). ( B ) Dose–response curves of PC-treated BMDM (left panel) and TR-treated BMDM (right panel). Data are expressed as a mean percentage of control growth ± Standard Deviation (SD) of two representative experiments ( n = 6 replicates per concentration).

Article Snippet: Primary human uterine fibroblast normal cells (HUF) were obtained from the ATCC.

Techniques: Derivative Assay, MTT Assay, Control, Standard Deviation, Concentration Assay

Journal: Allergy, asthma & immunology research

Article Title: Serum Amyloid A1: A Biomarker for Neutrophilic Airway Inflammation in Adult Asthmatic Patients.

doi: 10.4168/aair.2022.14.1.40

Figure Lengend Snippet: Fig. 1. The production of SAA1 from AECs and the effects of SAA1 on proinflammatory cytokine production. SAA1 proteins were produced from AECs (SAECs and A549, respectively) in response to various stimuli (A, B), and in the coculture with PBNs, PBEs (C, D). SAA1-induced release of IL-6, IL-8 and S100A9 from AECs (SAEC and A549, respectively) (E-K). Data are presented as means ± standard error of mean (n = 6). P values were calculated by using one-way analysis of variance with Bonferroni's post hoc test. SAA1 enhanced the expression of p-ERK and p-p38 MAPK (L) in AECs (SAECs and A549, respectively). IL, interleukin; PBEs, peripheral blood eosinophils; PBNs, peripheral blood neutrophils; dex, dexamethasone; S100A9, S100 calcium-binding protein A9; p-ERK, phospho-extracellular signal-regulated kinase; ERK, extracellular signal-regulated kinase; p-p38 MAPK, phospho-p38 mitogen-activated protein kinase; MAPK, mitogen-activated protein kinase; AECs, airway epithelial cells; SAEC, small airway epithelial cells. *P < 0.050, †P < 0.010, ‡P < 0.001, ns = not significant.

Article Snippet: Epithelial cell culture and stimulation Two kinds of AECs were used in this study, primary small airway epithelial cells (SAECs) and human AEC line (A549), both of which were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Produced, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by BPS

doi: 10.3390/ijms27073259

Figure Lengend Snippet: Representative high-content microscopy images of human coronary artery endothelial cells (HCAEC) exposed to vehicle control (CTRL) or 0.1 µM Bisphenol S (BPS) for 96 h and stained using the PhenoVue Cell Painting assay. For each condition, a representative field acquired at 40× magnification and a higher-magnification inset are shown. Rows correspond to the individual fluorescence channels: Hoechst 33342 (nuclei), PhenoVue Fluor 488 Concanavalin A (endoplasmic reticulum and intracellular membranes), PhenoVue 512 nucleic acid stain (RNA/nucleoli), PhenoVue Fluor 555 wheat germ agglutinin (plasma membrane), PhenoVue 641 mitochondrial stain (mitochondria), and the merged image. White boxes represent the part of the image used for the related inset. Scale bar: 50 µm, 40× objective.

Article Snippet: Primary human coronary artery endothelial cells (HCAEC; ATCC ® PCS-100-020TM, Innovation, VA, USA) were cultured according to the supplier’s recommendations.

Techniques: Microscopy, Control, Staining, Fluorescence, Clinical Proteomics, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection induces expression of a subset of YAP target genes. (A) Volcano plot of gene expression in bulk RNA-sequencing of End1/E6E7 immortalized epithelial cells (End1s) during infection with Chlamydia trachomatis ( Ct ) serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (B) Table of selected transcription factors identified as potential targets of infection-associated modulation via cross-referencing of differentially expressed genes identified in (A) with the ChIP Enrichment Analysis (ChEA) database of transcription factor target genes. See also Supplementary Data S2. (C) Volcano plot of gene expression in bulk RNA-sequencing of primary human endocervical epithelial cells (HCECs) during infection with Ct serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (D) Venn diagram of differentially expressed genes identified in (A, C) cross-referenced with the ChEA database of YAP target genes. (E) Scatter plot of gene expression of YAP-responsive (ChEA), differentially expressed genes in either Ct serovar L2-infected End1s (x-axis) or HCECs (y-axis). All fold changes are relative to each cell type’s respective mock-infected control; blue line: linear regression model of correlation; grey shading: 95% confidence interval. R 2 and p-values calculated using Pearson’s correlation. (F) Heatmap of YAP target gene expression in Ct serovar L2-infected End1s (left columns) and HCECs (right columns). All fold changes are relative to each cell type’s respective mock-infected control; only genes differentially expressed (FDRP ≤ 0.05) in both cell types are shown. (G) Dot plot of GO biological process term enrichment in the set of YAP target genes differentially expressed in Ct serovar L2 infection of End1s or HCECs (top 25 most significantly enriched terms shown). Dot size: number of term-associated genes found in set, dot color: adjusted p-value.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Expressing, Gene Expression, RNA Sequencing, Control, Targeted Gene Expression

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection promotes YAP nuclear translocation. (A) Expression of CTGF and CYR61 at 24 hpi in mock- and Ct L2-infected End1 cells, as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; n.s.: not significant (p-values > 0.05), asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (B) Expression of CTGF at 24 hpi in mock- and Ct L2-infected End1 cells transfected with non-targeting (NT) or YAP1-targeting siRNA (10 nM for 24 h prior to infection), as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (C) Expression of CTGF, INHBA, and BMP2 at 24 hpi in mock- and Ct L2-infected End1 cells treated with the YAP-TEAD inhibitor verteporfin (Vpf, 5 μM for 16 h starting at 8 hpi), as measured by RT-qPCR. n = 5 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (D) Representative micrographs of YAP (green) translocation into the nuclei (blue) of confluent mock- and Ct L2-infected End1 cells at 24 hpi. Asterisks: chlamydial inclusions, scale bar: 20 μm. (E) Quantification of YAP nuclear translocation in (D) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (F) Representative micrographs of YAP nuclear translocation of confluent mock- and Ct L2-infected primary human cervical epithelial cells at 24 hpi. Asterisks, chlamydial inclusions, scale bar: 20 μm. (G) Quantification of YAP nuclear translocation in (F) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (H) Quantification of YAP nuclear translocation at 2, 4, 8, 12, 18, and 24 hpi in confluent mock- and Ct L2-infected End1 cells as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Blue dots: mock-infected cells, red dots: Ct L2-infected cells, black bars: group means, asterisks: p-value ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons. (I) Representative micrographs of YAP nuclear translocation at 18 hpi in confluent mock- and Ct L2-infected End1 cells treated with chloramphenicol (Cm, 50 μg/mL for 1 h at 17 hpi) or DMSO. Asterisks: chlamydial inclusions; scale bar: 20 μm. (J) Quantification of YAP nuclear translocation in (I) . n = 3 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Translocation Assay, Expressing, Quantitative RT-PCR, Control, Transfection, Fluorescence

Journal: Allergy

Article Title: Human Pulmonary Neuroendocrine Cells Respond to House Dust Mite Extract With PAR-1 Dependent Release of CGRP.

doi: 10.1111/all.16416

Figure Lengend Snippet: FIGURE 1 | Characterization of iPNEC and ePNEC cultures. (A) Quantitative RT-PCR showing expression of characteristic PNEC markers in human cultured iPSC and -HBEC-derived iPNEC (□ female, 22; ■ male, 32) and ePNEC (● male, 52; ▲ male, 56; ○ female, 55) and control HBEC at day 60 in ALI culture, respectively. (B) Mean fluorescent intensity (MFI) of CHGA+/Hoechst+ cells (left) and SYP+/Hoechst+ cells [34] at day 60. Data is representative of four different culture plate wells per group for one biological donor for each cell type (ePNEC male, 52; iPNEC male, 32). (C) Representative IF images show SYP+ cells and nuclei are counterstained with Hoechst. 20× magnification and scale bars at 100 μm. (D) Single- cell RNA sequencing identifies two ePNEC clusters in 60-day-old ePNEC differentiated cells. Each dot represents one well and data shown for mean ± SEM. Mann–Whitney test performed for (A, B); * < 0.05. ASCL1, Achaete-Scute Family BHLH Transcription Factor 1; CHGA, Chromogranin A; ePNEC, epithelial-derived pulmonary neuroendocrine cells; iPNEC, iPSC-derived pulmonary neuroendocrine cells; iPSC, induced pluripotent stem cells; SYP, Synaptophysin.

Article Snippet: Cells were grown using the bronchial epithelial cell growth kit from ATCC (PCS300- 040).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Derivative Assay, Control, RNA Sequencing, MANN-WHITNEY

Journal: Allergy

Article Title: Human Pulmonary Neuroendocrine Cells Respond to House Dust Mite Extract With PAR-1 Dependent Release of CGRP.

doi: 10.1111/all.16416

Figure Lengend Snippet: FIGURE 4 | CGRP release after HDM stimulation is PAR1-dependent (A) Single-cell transcriptomics data of iPNECs (GSE146990, Hor et al.) and ePNEC for PAR1 (F2R), PAR2 (F2RL1), PAR3 (F2RL2), and PAR4 (F2RL3) genes. (B) SYP and PAR1 immunohistochemical co-staining in naive iPNEC (male, 32). 20× and 100× magnification and scale bars at 50 and 10 μm, respectively. (C, D) CALCB mRNA (C) and CGRP protein (D) expression in different ePNEC (● male, 52; ▲ male, 56; ○ female, 55) conditions after 2 h: 50 μL PBS, HDM (1200 μg/mL), PAR1 inhibitor Vorapaxar (80 μM) and PAR1 agonist TFLLR-NH2 (2 μM). (E) CALCB mRNA RT-PCR expression in different ePNEC conditions after 2 h: 50 μL PBS, HDM (1200 μg/mL), HDM co-incubation with protease inhibitors Chymostatin (broad spectrum, 10 μg/mL), PMSF (serine specific, 0.25 mM) and E-64 (cysteine specific, 10 μM). Inhibitor/agonist and or HDM added at the same time. Each dot represents one well and data shown for mean ± SEM. One-way ANOVA for (C, D, E); * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Symbol on top of multiple brackets represents all brackets. Agonist, PAR1 agonist vorapaxar; ePNEC, epithelial-derived pulmonary neuroendocrine cells; Inh., PAR1 inhibitor; iPNEC, iPSC-derived pulmonary neuroendocrine cells; HDM, House dust mite; PAR1, Protease activated receptor 1; SYP, Synaptophysin.

Article Snippet: Cells were grown using the bronchial epithelial cell growth kit from ATCC (PCS300- 040).

Techniques: Single-cell Transcriptomics, Immunohistochemical staining, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Derivative Assay