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: 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

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Western Blot analysis of emerin in primary mammary epithelial cells, MCF10A cells, MDA-231 cells and MDA-157 cells. (B) Western Blot quantification of emerin expression for each cell line. Emerin expression was normalized to tubulin and primary mammary epithelial cells. Error bars represent standard error. (n=3) ****p-value < 0.0001, unpaired t-test. (C) Nuclear area for MCF10A (n=116, blue), MDA-231 (n=237, green) and MDA-157 (n=342, red) cells. Error bars represent standard error. ****p-value < 0.0001, unpaired t-test (D) Representative DAPI (blue) images of MCF10A, MDA-231, and MDA-157 cells. Scale bars: 100 μm. (E) Nuclear circularity for MCF10A (n=75, blue), MDA-231 (n=301, green), and MDA-157 (n=237, red) cells. Error bars represent standard error. ****p-value < 0.0001, unpaired t-test

Article Snippet: Primary mammary epithelial cells were grown in mammary epithelial cell basal medium (ATCC, cat#: PCS-600–030) with all the components from the mammary epithelial cell growth kit (ATCC, cat#: PCS-600–040).

Techniques: Western Blot, Expressing

Journal: Nature communications

Article Title: LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation.

doi: 10.1038/s41467-018-08153-2

Figure Lengend Snippet: Fig. 5 LEADR regulates basal–luminal differentiation. a Bright-field images showing morphological changes occurring in RWPE-1 (left) and PrEC (right) cells (day 3) upon LEADR silencing by siLEADR (top) or gapLEADR (bottom). Scale bar, 50 µm. Full-size images reported in Supplementary Fig. 4c. b qRT-PCR showing changes in basal and luminal cytokeratins in RWPE-1 or PrEC cells upon LEADR silencing by siLEADR (top) or gapLEADR (bottom). Mean + s.d. plotted. c Western blots showing changes in basal/luminal cytokeratins in RWPE-1 (left) or PrEC (right) cells upon LEADR silencing by siLEADR (top) or gapLEADR (bottom). Vinculin used as loading control. d Immunofluorescence showing cytoplasmic re-localization of p63 (green) upon LEADR silencing in RWPE-1 cells by siLEADR (top) or gapLEADR (bottom). Nuclei counterstained with DAPI (blue). Scale bar, 50 µm. Full-size images are reported in Supplementary Fig. 4d. e Western blots showing changes in Androgen Receptor (AR) expression in PrEC cells upon LEADR silencing by siLEADR (left) or gapLEADR (right). GAPDH used as loading control. f Immunofluorescence showing AR (red) expression in PrEC cells upon LEADR silencing by siLEADR, in the presence or absence of simultaneous DHT stimulation. Nuclei counterstained with DAPI (blue). Scale bar, 50 µm. Full-size images reported in Supplementary Fig. 4g. g ELISA-based quantification of PSA in the conditioned media of PrEC cells silenced for LEADR expression by siLEADR (±DHT). Mean + s.d. (n = 2) plotted. h Heatmap (bottom) of the normalized enrichment score (NES) for luminal and basal custom gene sets (obtained from gene- expression data of frankly basal and luminal prostate cells from the indicated datasets) in RWPE-1 cells upon knockdown of LEADR and/or miR-205 (the latter abrogated using an antisense LNA-modified oligomer, LNA205). Bar plot of differential luminal/basal NES is reported (top). Mean + s.d. (n = 4) plotted. i Plot showing propensity of wild type (wt), LEADR-overexpressing and CRISPRed RWPE-1 cells to differentiate toward luminal phenotype (measured by qRT-PCR and expressed as KRT18/KRT5 ratio) upon culturing in media with increasing differentiative potential (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (Student’s t test). Source data are provided as a Source Data file, together with n of all experiments

Article Snippet: The normal primary prostate epithelial cells (PrEC) were grown in PrEC basal medium (ATCC; PCS-440-030) supplemented with PrEC growth kit (ATCC; PCS-440-040).

Techniques: Quantitative RT-PCR, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression, Knockdown

Journal: Frontiers in pharmacology

Article Title: The Anticancer Effects of the Pro-Apoptotic Benzofuran-Isatin Conjugate (5a) Are Associated With p53 Upregulation and Enhancement of Conventional Chemotherapeutic Drug Efficiency in Colorectal Cancer Cell Lines.

doi: 10.3389/fphar.2022.923398

Figure Lengend Snippet: FIGURE 7 | Compound 5a decreases HT29 and SW620 cell viability without affecting the viability of normal colon epithelial CCD 841 cells and enhances irinotecan (IRI), 5-fluorouracil (5-FU), and oxaliplatin (OXA) cytotoxic effects on HT29 and SW620 cells. (A) HT29, SW620, and the normal colon epithelial CCD 841 cell lines were exposed to different concentrations (5-10-20 μM) of Compound 5a for 24 h. Cell viability was measured by the MTT assay at 540 nm regarding the cellular metabolic activity. Bar graph showing the cell viability percentage and the data are expressed as mean ± SD (n = 3). ***p < 0.001 and ****p < 0.0001 vs. Control. Half-maximal inhibitory concentrations (IC50) of Compound 5a on HT29 and SW620 cell viability were determined. HT29 (B,C) and SW620 (D,E) cells were treated with different concentrations of the chemotherapeutic drugs IRI, 5-FU, and OXA for 24 h in the presence (C,E) or absence (B,D) of various concentrations (5-10-20 μM) of Compound 5a. Cell cytotoxicity was measured by the MTT assay at 540 nm. Bar graph showing the cell viability percentage and the data are expressed as mean ± SD (n = 3). Half- maximal inhibitory concentrations (IC50) of each chemotherapeutic drug on HT29 and SW620 cell viability were also determined.

Article Snippet: Human normal colon epithelial cell (CCD841 CoTr), colorectal adenocarcinoma HT29, and mCRC SW620 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States) and grown in a complete medium composed of DMEM supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 IU/ml penicillin and 2 mmol/l L-glutamine.

Techniques: MTT Assay, Activity Assay, Control