cell culture adenocarcinomas human alveolar basal epithelial cells line  (ATCC)

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 1. Protein expression of the 5-lipoxygenase pathway and the BLT1 receptor in A549 non-small lung cancer cells. (A) mRNA levels of cPLA2 , 5-LOX, FLAP, LTA4 H and BLT1 normalized with housekeeping (pumilio) in the cell line. (B) The protein lysate from A549 cells were analyzed by immunoblot and the membranes were incubated with primary antibodies against cPLA2 , 5-LOX, FLAP, LTA4 H, BLT1 and β-actin. ( C) Semi-quantification of the relative protein expression of cPLA2 , 5-LOX, FLAP, LTA4 H and BLT1, normalized with β-actin. The values 110, 78, 15, 70, 37, and 43 correspond to mass units (kDa). cPLA2 , 5-LOX, LTA4 H, BLT1; n = 3; FLAP; n = 4

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Expressing, Western Blot, Incubation

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 3. Effect of ALA on LTB4 secretion in A549 non-small lung cancer cells. Cells were pre-incubated with SC (0.01 μM), ALA (0.01 μM), or ALA (10 μM) for 30 min- utes, and then stimulated with PMA (5 nM) for 15 minutes. LTB4 secretion was determined by ELISA Kit. Data are presented as the median with interquartile range (n = 3 independent experiments; ∗P ≤0.05; Kruskal-Wallis Test).

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 4. Effect of LTB4 on the expression of canonical markers of epithelial mesenchymal transition in non-small cell lung cancer A549. (A) The cells were incubated with IL-6 or different concentrations of LTB4 for 48 hours, subsequently the protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against E-cadherin, Vimentin, ZEB-1, and b-actin. (B) Semi-quantification of the relative protein expression of E-cadherin normalized with β-actin and the control. (C) Semi-quantification of the relative protein expression of Vimentin normalized with β-actin and the control. (D) Semi-quantification of the relative protein expression of ZEB-1 normalized with β-actin and the control. The values 100, 55, 180, and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (ZEB-1 (n = 3); Vimentin and E-cadherin (n = 4) independent experiments; ∗∗P ≤0.01; Kruskal-Wallis Test).

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Expressing, Incubation, Western Blot, Control

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 5. Effect of ALA-mediated LTB4 decrease on the expression of canonical markers of epithelial mesenchymal transition in non-small cell lung cancer A549. (A) Cells were pre-incubated with SC (0.01 μM) or ALA (0.01 or 10 μM) for 30 minutes, and then stimulated with PMA (5 nM), for 15 minutes, LTB4 (100 nM), 50 ng/mL IL-6 or IL- 6/LTB4 for 48 hours, subsequently the protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against E-cadherin, Vimentin, ZEB-1 and β-actin. (B) Semi-quantification of the relative protein expression of E-cadherin normalized with β-actin and the control. (C) Semi-quantification of the relative protein expression of Vimentin normalized with β-actin and the control. (D) Semi-quantification of the relative protein expression of ZEB-1 normalized with β-actin and the control. The values 70, 55, 180, and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (n = 3 independent experiments; ∗P ≤ 0.05; Kruskal-Wallis Test).

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Expressing, Incubation, Western Blot, Control

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 6. Effect of LTB4 and ALA-mediated LTB4 decrease on the viability and proliferation in non-small cell lung cancer A549. ( A) The cells were stimulated with different concentrations of LTB4 for 12 hours, subsequently the percentage of viable cells was evaluated through MTT. Cells were pre-incubated with SC (0.01 μM) or ALA (0.01 or 10 μM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. (B) Percentage of viable cells by MTT. (C) Protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against PCNA and β-actin. (D) Semi-quantification of the relative protein expression of PCNA normalized with β-actin and the control. The values 36 and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (viability (n = 3); proliferation (n = 4) independent experiments; ∗P ≤0.05 and ∗∗P ≤0.01; Kruskal-Wallis Test).

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Incubation, Western Blot, Expressing, Control

Journal: Current Therapeutic Research

Article Title: Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

doi: 10.1016/j.curtheres.2024.100765

Figure Lengend Snippet: Figure 7. Effect of ALA-mediated LTB4 decrease on invasive capacities, motility, and colony formation in non-small cell lung cancer A549. Cells were pre-incubated with SC (0.01 μM) or ALA (0.01 or 10 μM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. (A) Illustration of the migration transwell test per 6 hours, Scale bar; 1 mm. (B) Number of cells migrated per 6 hours. (C) Illustration of the invasion transwell test per 20 hours, Scale bar; 1 mm. (D) Number of cells invaded per 20 hours. (E) Illustration of the colony formation assay after 6 days, Scale bar; 1 cm and 1 mm (zoom). (F) Quantification of the number of colonies after 6 days. (G) Illustration of the colony formation test at 9 days, Scale bar; 1 cm and 1 mm (zoom). (H) Quantification of the number of colonies after 9 days. Data are presented as the median with interquartile range (migration and invasion n = 3 independent experiments; ∗P ≤0.05; Kruskal-Wallis Test). colony formation (6 days; n = 3 independent experiments; ∗P ≤ 0.05; Mann-Whitney and 9 days; n = 4 independent experiments; ∗P ≤0.05; Mann-Whitney).

Article Snippet: The alts, alcohols (ethanol and isopropanol), chloroform and dimethylulfoxide were acquired from Winkler, Ltd (Lampa, Santiago, Chile) ethods ell culture Adenocarcinomic human alveolar basal epithelial cells (A549) ere purchased from the American Type Culture Collection (ATCC, aithersburg, Maryland).

Techniques: Incubation, Migration, Colony Assay, MANN-WHITNEY

Journal: MedComm

Article Title: Pyrogallol protects against influenza A virus‐triggered lethal lung injury by activating the Nrf2–PPAR‐γ–HO‐1 signaling axis

doi: 10.1002/mco2.531

Figure Lengend Snippet: Effects of Nrf2–PPAR‐γ–HO‐1 signal axis inhibition on H1N1 virus‐activated RIG‐I–NF‐κB signaling in A549 cells with pyrogallol treatment. (A) The cytotoxicity of pyrogallol. (B) Immunoblot analysis of Nrf2 expression. (C) Immunoblot analysis of HO‐1 expression. (D and E) Levels of ROS. (F) GSH/GSSG ratio. (G) Immunoblot analysis of PPAR‐γ in A549 cells infected with the H1N1 virus and treated with pyrogallol. (H) Immunoblot analysis of PPAR‐γ and HO‐1 expression. (I) Immunoblot analysis of HO‐1 expression. (J) Immunoblot analysis of RIG‐I–NF‐κB signaling activation. (K) The Luminex assay was implemented to measure the levels of proinflammatory mediators (IL‐6, IL‐8, IP‐10, MCP‐1, RANTES, and TNF‐α) in the culture supernatant. (L) Four‐color immunofluorescence staining to measure the expression of proinflammatory mediators (IL‐6, TNF‐α, IL‐8, and MCP‐1) in SpC + alveolar epithelial cells. (M) Panel representation of the relative fluorescence intensity of proinflammatory cytokines. Data were normalized to the control group. (N) Four‐color immunofluorescence staining to measure the expression of p‐p65 (pink) and HO‐1 (red) in SpC + (green) alveolar epithelial cells. (O) Panel representation of the relative fluorescence intensity of p‐p65 and HO‐1. Data were normalized to the control group. (P) Expression of RIG‐I was examined by immunoblotting. (Q) Immunoblot analysis of RIG‐I, p‐IKBα, and p‐p65 expression. (R) Luminex assay was implemented to measure the levels of proinflammatory mediators (IL‐6, IL‐8, and IP‐10) in the culture supernatant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: DMEM/F12 with 10% (v/v) fetal bovine serum was used to culture human A549 alveolar epithelial cells (ATCC; CCL‐185) at 37°C in a humidified environment with 5% CO 2 .

Techniques: Inhibition, Virus, Western Blot, Expressing, Infection, Activation Assay, Luminex, Immunofluorescence, Staining, Fluorescence, Control

Journal: MedComm

Article Title: Pyrogallol protects against influenza A virus‐triggered lethal lung injury by activating the Nrf2–PPAR‐γ–HO‐1 signaling axis

doi: 10.1002/mco2.531

Figure Lengend Snippet: Effects of pyrogallol on H1N1 virus‐triggered apoptosis in vitro and in vivo. (A) Flow cytometry was used to identify the apoptotic state of A549 cells infected with the H1N1 virus. (B) The percentage of cells that have undergone apoptosis in panel (A). (C) Expression of cleaved caspase 3 and cleaved PARP as determined by immunoblotting. (D) Levels of TRAIL were measured by the Luminex assay. (E) Four‐color immunofluorescence staining to examine the apoptosis of SpC + (pink) alveolar epithelial cells (red, active caspase 3; green, TUNEL) in the lungs. (F) Panel showing the relative fluorescence intensity of active caspase 3 and TUNEL. Data were normalized to the control group. (G) Immunoblot analysis of FTH‐1, SLC7A11, and GPX4 expression. (H) Immunoblot analysis of p‐MLKL, MLKL, RIPK1, and RIPK3 expression. (I) Flow cytometry was used to identify the apoptotic state of A549 cells infected with the H1N1 virus. (J) The percentage of apoptotic A549 cells infected with the H1N1 virus in panel (I). (K) Expression of cleaved caspase 3 and cleaved PARP as determined by immunoblotting. (L) The Luminex assay was used to assess the levels of TRAIL. (M) Immunoblot analysis of FTH‐1, SLC7A11, and GPX4 expression. (N) Immunoblot analysis of p‐MLKL, MLKL, RIPK1, and RIPK3 expression. (O) TEM analysis of the cellular ultrastructure. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: DMEM/F12 with 10% (v/v) fetal bovine serum was used to culture human A549 alveolar epithelial cells (ATCC; CCL‐185) at 37°C in a humidified environment with 5% CO 2 .

Techniques: Virus, In Vitro, In Vivo, Flow Cytometry, Infection, Expressing, Western Blot, Luminex, Immunofluorescence, Staining, TUNEL Assay, Fluorescence, Control

Journal: MedComm

Article Title: Pyrogallol protects against influenza A virus‐triggered lethal lung injury by activating the Nrf2–PPAR‐γ–HO‐1 signaling axis

doi: 10.1002/mco2.531

Figure Lengend Snippet: Effects of pyrogallol on H1N1 virus‐elicited amplification of the proinflammatory response in IFN‐β‐pretreated cells. (A and B) The concentration of IFN‐β in the cell culture supernatant of A549 cells infected with the H1N1 virus (A) and vRNA‐transfected A549 cells (B). (C) Luminex assay was implemented to measure the levels of proinflammatory mediators (IL‐6, IL‐8, IP‐10, MCP‐1, MIP‐1α, and TNF‐α) in the culture supernatant. (D) Flow cytometry was used to determine the apoptosis of H1N1 virus‐infected A549 cells that had been pretreated with IFN‐β. (E) The proportion of apoptotic cells in panel (D). (F) Levels of TRAIL were measured by Luminex assay. (G) The ISRE‐luciferase reporter activity was measured in H1N1 virus‐infected A549 cells. (H) ISRE‐luciferase reporter activity in A549 cells pretreated with IFN‐β (500 ng/mL) before H1N1 virus infection. (I) Immunoblotting of p‐JAK1, p‐STAT1, and p‐STAT2 expression in A549 cells stimulated for 15 min with IFN‐β (20 ng/mL). (J) Immunofluorescence analysis was conducted to detect the nuclear localization of p‐STAT1 and p‐STAT2 in A549 cells stimulated for 15 min with IFN‐β (20 ng/mL). (K) Immunoblotting of p‐JAK1, p‐STAT1, and p‐STAT2 expression in A549 cells infected with H1N1 virus for 4 h followed by 15 min of stimulation with IFN‐β (20 ng/mL). (L) The expression of RIG‐I in A549 cells was examined by immunoblotting after 24 h of stimulation with IFN‐β (500 ng/mL). (M) Expression of RIG‐I in A549 cells treated with IFN‐β (500 ng/mL) for 4 h before H1N1 virus infection was examined by immunoblotting. (N) A549 cells transfected with the RIG‐IOE plasmid were analyzed by immunoblotting for the expression of p‐STAT1 and p‐STAT2. (O) H1N1 virus infection of RIG‐I OE plasmid‐transfected A549 cells for 8 h was analyzed by immunoblotting for p‐STAT1 and p‐STAT2 expression. (P) Immunoblot analysis of p‐STAT1 and p‐STAT2 expression in A549 cells with vRNA transfection. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: DMEM/F12 with 10% (v/v) fetal bovine serum was used to culture human A549 alveolar epithelial cells (ATCC; CCL‐185) at 37°C in a humidified environment with 5% CO 2 .

Techniques: Virus, Amplification, Concentration Assay, Cell Culture, Infection, Transfection, Luminex, Flow Cytometry, Luciferase, Activity Assay, Western Blot, Expressing, Immunofluorescence, Plasmid Preparation

Journal: MedComm

Article Title: Pyrogallol protects against influenza A virus‐triggered lethal lung injury by activating the Nrf2–PPAR‐γ–HO‐1 signaling axis

doi: 10.1002/mco2.531

Figure Lengend Snippet: Involvement of HO‐1 in the inhibitory effect of pyrogallol on IFN‐β‐mediated amplification of the proinflammatory response and exacerbated apoptosis in cells with H1N1 virus infection. (A) Expression of HO‐1 in A549 cells treated with IFN‐β (500 ng/mL) for 4 h before H1N1 virus infection was examined by immunoblotting. (B) Levels of proinflammatory mediators (IL‐6, TNF‐α, and IP‐10) in A549 cells stimulated with IFN‐β (500 ng/mL) for 4 h before H1N1 virus infection. (C) Flow cytometry was used to assess the apoptosis of H1N1 virus‐infected A549 cells that had been pretreated with IFN‐β. (D) The proportion of apoptotic cells in panel (C). (E) The levels of TRAIL were measured by the Luminex assay. (F) Expression of p‐JAK1, p‐STAT1, and p‐STAT2 in A549 cells treated with IFN‐β (20 ng/mL) for 15 min was analyzed by immunoblotting. (G) Expression of p‐JAK1, p‐STAT1, and p‐STAT2 in A549 cells stimulated with IFN‐β (20 ng/mL) for 15 min after infection with H1N1 virus for 4 h was analyzed by immunoblotting. (H) Analysis of the expression of RIG‐I by immunoblotting in A549 cells treated with IFN‐β (500 ng/mL) for 24 h. (I) Evaluation of RIG‐I expression by immunoblotting in A549 cells treated with IFN‐β (500 ng/mL) for 4 h before infection with the H1N1 virus. (J) Assessment of p‐STAT1 and p‐STAT2 expression in A549 cells transfected with the RIG‐I OE plasmid using immunoblotting. (K) Expression of p‐STAT1 and p‐STAT2 in A549 cells transfected with the RIG‐I OE plasmid and infected for 8 h with the H1N1 virus was analyzed by immunoblotting. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: DMEM/F12 with 10% (v/v) fetal bovine serum was used to culture human A549 alveolar epithelial cells (ATCC; CCL‐185) at 37°C in a humidified environment with 5% CO 2 .

Techniques: Amplification, Virus, Infection, Expressing, Western Blot, Flow Cytometry, Luminex, Transfection, Plasmid Preparation