human lung alveolar epithelial cell line a549  (DSMZ)

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: The in vitro stimulation of lung epithelial cells, macrophages, and neutrophil-precursor cells. Depicted are flow cytometric analyses showing CD69+ ( a – c )- and TLR4+ ( d – f )-stimulated A549, THP1 and HL60 cells. The activation of the NF-kB pathway was detected via a Western blot in all three cell lines, showing the comparison of the NF-kB p50 band in unstimulated and stimulated cells at 50 kDa. YY1 is used as an endogenous control for nuclear protein expression ( g – i ). Further, IL-8 protein expression levels were measured by a cytometric bead array (CBA) after cytokine mix (CM4, CM6, and CM8) stimulation in A549 ( j ) and in HL60 cells ( l ), as well as after M1 differentiation in THP1 cells ( k ). A549: n = 4; THP1: n = 3–8; HL60: n = 3–7. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: In Vitro, Activation Assay, Western Blot, Comparison, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: Analysis of miRNA expression in stimulated cell lines. Volcano plots illustrate differentially expressed miRNAs measured by next generation sequencing (NGS) in CM8-stimulated A549 cells ( a ), M1 THP1 cells ( b ), and CM8-stimulated HL60 cells ( c ) compared to unstimulated or M0 THP1 cells, respectively ( n = 4). Green dots represent significantly upregulated miRNAs (log2 fold change (FC) > 1; padj < 0.001) and red dots represent significant downregulation of miRNA expression (log2 FC < −1; padj < 0.001). Vertical dotted lines in the volcano plots demonstrate the thresholds of fold changes for further analyses. Venn blot analysis (Venny 2.1) shows comparison of significantly upregulated miRNAs with padj < 0.001 and log2 FC > 1 in stimulated A549, THP1, and HL60 cells ( d ).

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: Expressing, Next-Generation Sequencing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: Effects of miRNA transfection on IL-8 mRNA and protein expression. Stimulated A549, THP1, and HL60 cells were transfected with mimics and inhibitors of miR-146a-5p ( a – f ) and miR-146a-3p ( g – l ). IL-8 mRNA expression was measured by qPCR in CM8-stimulated A549 cells ( a , g ), in M1 THP1 cells ( b , h ), and in CM8-stimulated HL60 cells ( c , i ). Protein expression was measured by CBA in supernatants of cell culture samples ( d – f , j – l ). FC = fold change; NC = negative control. A549: n = 6–12, THP1: n = 3–5, HL60: n = 3–6, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: Transfection, Expressing, Cell Culture, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: The impact of the simultaneous modification of miR-146a-5p and miR-146a-3p on cytokine expression. The mimic of miR-146a-5p and the inhibitor of miR-146a-3p were transfected separately and in combination in stimulated A549 ( a , b ) and THP1 cells ( c ), measuring IL-8 ( a , c ) and IL-6 protein expression ( b ) in the supernatant of cell culture samples. The reverse combination consisting of transfection with the miR-146a-5p inhibitor and miR-146a-3p mimics was applied in ( d – f ). A549: n = 5–6, THP1: n = 3; * p < 0.05, ** p < 0.01.

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: Modification, Expressing, Transfection, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: Target analysis of miR-146a-5p and miR-146a-3p. In silico target analysis was performed using www.mirnet.ca showing 203 targets of miR-146a-5p and 116 targets of miR-146a-3p including three overlapping targets. KEGG pathway analysis demonstrates 15 targets implicated in Toll-like receptor signaling (yellow), eight genes involved in RIG-I-like receptor signaling (green), and 12 targets belonging to chemokine signaling pathway (orange) ( a ). Targets that are allocated to multiple pathways are shown as increased dots. Pathways found in the KEGG pathway analysis and their respective padj values are displayed in ( b ) with relevant pathways colored in red. Effects of target modulation by miR-146a-5p mimic and anti-miR-146a-5p transfection were measured individually and in combination in stimulated A549 cells. qPCR measurements show expression of IRAK1 ( c ), TRAF6 ( d ), DDX3X ( e ), RNF125 ( f ), and CXCR4 ( g ) in transfected cells. n = 3. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: In Silico, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases

doi: 10.3390/ijms25147686

Figure Lengend Snippet: Mutual interference of miR-146a-5p and miR-146a-3p expression in vitro. miR-146a-3p expression was measured by qPCR after transfection with miR-146a-5p mimic in stimulated A549 cells ( a ), in M1 THP1 cells ( b ), and in stimulated HL60 cells ( c ). miR-146a-5p expression after transfection with miR-146a-3p mimic in the respective cell lines is depicted in ( d – f ). A549: n = 4–6, THP1: n = 6–7, HL60: n = 3–5. * p < 0.05, **** p < 0.0001.

Article Snippet: Human lung alveolar epithelial cell line A549 was obtained from DSMZ and was maintained in a DMEM medium (ThermoFisher, Dreieich, Germany) containing 10% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin–streptomycin (Life Technologies, Darmstadt, Germany) at a density of 2 × 10 6 cells per T75 cell culture flask.

Techniques: Expressing, In Vitro, Transfection

Journal: Nature Communications

Article Title: Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins

doi: 10.1038/s41467-024-47741-3

Figure Lengend Snippet: a IC 50 values of PB-1 , PB-2 , and PB-3 (distinct samples n = 3, 95% CI for IC 50 and error bars represent ± S.D.) corresponding to the ability of PB-inhibitors to block the PLY-induced hemoglobin release in sheep erythrocytes. b PLY was inhibited by PB-3 , formed via protein-templated fragment ligation in the hemolysis assay, while PB-3.1 and PB-2.3 alone were no inhibitors (distinct samples n = 3, error bars represent ± S.D.). c Quantification of PB-3 formed by protein-templated ligation using HPLC-QTOF-MS. Concentrations were fitted to the one-phase saturation equation c(t) = c 0 + ( c max - c 0 )*(1-exp(-K*t)) used to obtain c max (795 nM) and t 1/2 (~20.5 min) (distinct samples n = 3, error bars represent ± S.D.). d , e PB-3 prevents human alveolar epithelial cells from PLY-associated impairment. Samples include controls (positive and negative), PLY alone, and PLY with PB-3 (1, 3 and 6 µM). Figures show LDH release (which is quantified as percent-cytotoxicity) after 4 h and 24 h. Mann Whitney test (two-tailed) is used for statistical evaluation (distinct samples n = 8, *** p = 0.0009 for d distinct samples n = 8 *** p = 0.0002 for ( e ), and error bars represent ± S.D.). f Binding kinetics of PB-3 and PLY complex measured in the BLI assay. PLY-C428A is used as reference protein and buffer is reserved as inhibitor reference. Binding is observed over 0.6, 1, and 6 µM of PB-3 (data shown are representing one of n = 3 distinct samples). The reference-subtracted data are presented in the graph, which display association and dissociation steps of wild-type PLY and PB-3 . g The one-to-one kinetic fitting model was used to calculate the on- and off-rates ( k on , k off ) of binding and the binding affinity ( K D ) of PB-3 to PLY.

Article Snippet: The human lung alveolar epithelial cell line A549 (ATCC, CCL-185) was cultured in Ham’s F12 medium (Biochrome, Berlin, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 2 mM L-glutamine (Invitrogen Life Technologies) at 37 °C and 5% CO2.

Techniques: Blocking Assay, Ligation, Hemolysis Assay, MANN-WHITNEY, Two Tailed Test, Binding Assay

Journal: Nature Communications

Article Title: Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins

doi: 10.1038/s41467-024-47741-3

Figure Lengend Snippet: PLY neutralized by PB-3 in human alveolar epithelial cells (A549 cells). Cells were grown in Ham’s F12 culture medium with inclusion of a red mitochondrial dye (TMRE: tetramethyl-rhodamine, ethyl ester) to indicate healthy cells and a fluorogenic peptide substrate (DEVD) with green fluorescence to highlight activity of caspase 3 and 7 activity in apoptotic cells (data here represent one of n = 3, distinct samples). a (I) Healthy cells as a negative control, 24 h; (II) cells treated with PLY 15 nM, 24 h; (III) cells treated with PLY (15 nM) + PB-3 (6 µM), 24 h; (IV) cells with PB-3 as a control (24 h). b PB-3 blocked cellular deterioration in A549 cells infected with S.pn . D39 strain (data shown is one of n = 3, distinct samples). (I) healthy control cells, (II) cells infected with D39Δcps induced cellular injury (16 h), (III) cells infected with D39Δcps treated with PB-3 (6 µM) 4-times after regular intervals of 4 h (16 h), and (IV) D39ΔcpsΔply control, and it represents that only PLY provokes the cellular injury. All images were recorded by laser confocal microscopy after the mentioned time intervals. Scale bars correspond to 50 µm.

Article Snippet: The human lung alveolar epithelial cell line A549 (ATCC, CCL-185) was cultured in Ham’s F12 medium (Biochrome, Berlin, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 2 mM L-glutamine (Invitrogen Life Technologies) at 37 °C and 5% CO2.

Techniques: Fluorescence, Activity Assay, Negative Control, Control, Infection, Confocal Microscopy