a549 human type ii alveolar cells  (ATCC)

Journal: mBio

Article Title: Pneumococcal H₂O₂ reshapes mitochondrial function and reprograms host cell metabolism

doi: 10.1128/mbio.02019-25

Figure Lengend Snippet: Spn-H 2 O 2 modifies mitochondrial morphology, induces aggregation, and promotes mislocalization. Human alveolar A549 cells were either mock-infected or infected with Spn strains and incubated for 8 h at 37°C with 5% CO 2 . Infected cells were stained for mitochondria (green), DNA (blue), and actin (red), imaged via confocal microscopy, and analyzed using Imaris software. Panels ( A and B ) display XY optical-middle sections (0.5 µm) from z-stacks. Panel ( B ) additionally includes XY, XZ (bottom), and YZ (side) optical sections. Three-dimensional reconstructions were generated from z-stacks, with panel ( C ) presenting simultaneous XY and XZ optical sections. Arrows in panels ( B, C ) indicate aggregated mitochondria. Confocal microscopy images are representative of at least three independent experiments. ( D–G ) Imaris software was employed to generate masks from the mitochondrial channel, enabling the assessment of subcellular localization (apical, middle, or basal) and quantification of mitochondrial area (µm²). Panel ( D ) depicts cells infected with strain EF3030, with the top panel showing all z-stacks, highlighting predominant apical mislocalization of mitochondria, and the bottom panel a middle optical section. Panel ( E ) presents three-dimensional images across all conditions as in ( D ), while panel ( F and G ) quantifies mitochondrial area under each infection condition. Data are expressed as mean ± SEM from confocal micrographs of three independent experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s post hoc test, and **** indicating P < 0.0001.

Article Snippet: A549 human type II alveolar epithelial cells (ATCC, CCL-185) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gibco), and 100 U/mL of penicillin-streptomycin (Gibco) in 25 cm 2 flasks (Fisher).

Techniques: Infection, Incubation, Staining, Confocal Microscopy, Software, Generated

Journal: mBio

Article Title: Pneumococcal H₂O₂ reshapes mitochondrial function and reprograms host cell metabolism

doi: 10.1128/mbio.02019-25

Figure Lengend Snippet: Streptococcus pneumoniae -derived H₂O₂ does not significantly alter mitochondrial membrane potential in lung epithelial cells. ( A–H ) Human alveolar A549 cells were mock-treated, treated with CCCP, H 2 O 2 , EF3030, EF3030Δ spxB Δ lctO, or EF3030 and CCCP for 10 h. Cells were stained with JC-1 dye for flow cytometric analysis and counterstained with SYTOX Blue to exclude dead cells. ( A–F ) Scatter plots showing JC-1 fluorescence (PE-TexasRed/FITC) for each condition, with gating to identify live cells. ( G and I ) Quantification of change in mitochondrial membrane potential (Δψm) or ( H and J ) percent of live cells expressed as mean ± SEM from three independent experiments; one-way ANOVA with Dunnett’s post hoc test; ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: A549 human type II alveolar epithelial cells (ATCC, CCL-185) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gibco), and 100 U/mL of penicillin-streptomycin (Gibco) in 25 cm 2 flasks (Fisher).

Techniques: Derivative Assay, Membrane, Staining, Fluorescence

Journal: mBio

Article Title: Pneumococcal H₂O₂ reshapes mitochondrial function and reprograms host cell metabolism

doi: 10.1128/mbio.02019-25

Figure Lengend Snippet: Limited contribution of Spn-H 2 O 2 to apoptosis in lung epithelial cells. ( A ) Human alveolar A549 cells were mock-infected, treated with DNaseI (1U), treated with H 2 O 2 (2 mM) added every 2 h, or infected with TIGR4, TIGR4Δ spxB Δ lctO , EF3030, or EF3030Δ spxB Δ lctO for 10 h. Cells were stained with the Click-iT Plus TUNEL assay kit, and mounted on slides with DAPI-containing resin and analyzed by confocal microscopy using the Imaris software. ( B ) Fluorescence intensity (FITC/GFP) from each condition, reported as arbitrary units (AU), quantifying TUNEL staining. ( C ) A549 cells were mock-infected or infected with EF3030, EF3030Δ spxB Δ lctO for 10 h or treated with staurosporine (10 µM, 4 h), then stained with Cell Event Caspase-3/7 Green Detection Reagent (2 µM) and SYTOX Blue Dead Cell Stain (1 µM) prior to flow cytometric analysis. Representative bivariate density plots show the percentages of single A549 cells within the P3 gate: non-apoptotic SYTOX blue–positive cells (Q4-1), SYTOX blue–negative non-apoptotic cells (Q4-3), and caspase-3/7–positive, SYTOX blue–negative cells (Q4-4) for each condition. ( D ) Percentage of SYTOX Blue-negative live cells, and among those, the proportion of caspase-3/7-positive cells. ( B, D ) Data represent mean ± SEM from two independent experiments with four ( B ) or two ( D ) internal replicates each; one-way ANOVA with Dunnett’s post hoc test; ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001. ( E, F ) Cells treated with staurosporine (10 µM) for 7 h or infected with EF3030 for 10 h were stained with WGA, caspase-3/7 reagent, and DAPI as indicated. Z-stacks were acquired in real time using confocal microscopy at the specified time points. Displayed are the middle sections of 3D images from z-stacks (left panels) and the corresponding XY, XZ, and YZ optical sections (right panels). ( G, H ) RNA-seq analysis of Calu-3 ( G ) or BEAS-2B ( H ) cells mock-infected or infected for 9 or 16 h with Spn strains. Heatmaps depict the expression of mitochondria-related and apoptosis-related genes.

Article Snippet: A549 human type II alveolar epithelial cells (ATCC, CCL-185) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gibco), and 100 U/mL of penicillin-streptomycin (Gibco) in 25 cm 2 flasks (Fisher).

Techniques: Infection, Staining, TUNEL Assay, Confocal Microscopy, Software, Fluorescence, RNA Sequencing, Expressing

Journal: Current Issues in Molecular Biology

Article Title: miR-7704-Enriched Stem Cell-Derived Extracellular Vesicles Attenuate Hyperoxia-Induced Apoptosis and Oxidation in Lung Epithelial Cells

doi: 10.3390/cimb47110893

Figure Lengend Snippet: Uptake of EVs by A549 Cells. EVs were labeled with red fluorescence using the ExoGlowTM-Membrane EV Labeling Kit. Fluorescence microscopy images showing uptake of red-labeled exosomes by A549 cells, with nuclei stained blue by Hoechst H33342 and merged images indicating intracellular localization. Magnification: ( A – C ) 50×, scale bar = 100 μm, ( D – F ) 200×, scale bar = 100 μm, and ( G – L ) 400×, scale bar = 50 μm.

Article Snippet: After thawing in liquid nitrogen, human type II alveolar epithelial A549 cells (ATCC CCL-185, Manassas, VA, USA) were cultured in F-12K Nutrient Mixture (Gibco, Waltham, MA, USA) containing 10% FBS and 1% penicillin/streptomycin for three passages.

Techniques: Labeling, Fluorescence, Membrane, Microscopy, Staining

Journal: Current Issues in Molecular Biology

Article Title: miR-7704-Enriched Stem Cell-Derived Extracellular Vesicles Attenuate Hyperoxia-Induced Apoptosis and Oxidation in Lung Epithelial Cells

doi: 10.3390/cimb47110893

Figure Lengend Snippet: Effects of miR-7704-enriched EVs on A549 cell proliferation under hyperoxic conditions. ( A ) Cell viability of A549 cells cultured under normoxia or 85% hyperoxia for 0–72 h, treated with control medium, HUCMSC-derived EVs, or miR-7704-enriched HUCMSC EVs, was measured by XTT assay. ( B ) Growth curves indicate that hyperoxia significantly suppresses cell proliferation, whereas treatment with miR-7704-EVs partially restores cell viability compared to the hyperoxia control group. ( C ) A statistical comparison of OD values at 48 and 72 h revealed significant differences between the hyperoxia control and normoxia groups, as well as between the hyperoxia control and HUCMSC-EV-treated groups. In contrast, miR-7704-EV treatment showed attenuated hyperoxia-induced growth inhibition. Data are presented as the mean ± standard deviation (SD) ( n = 3); p < 0.05 was considered statistically significant.

Article Snippet: After thawing in liquid nitrogen, human type II alveolar epithelial A549 cells (ATCC CCL-185, Manassas, VA, USA) were cultured in F-12K Nutrient Mixture (Gibco, Waltham, MA, USA) containing 10% FBS and 1% penicillin/streptomycin for three passages.

Techniques: Cell Culture, Control, Derivative Assay, XTT Assay, Comparison, Inhibition, Standard Deviation

Journal: Current Issues in Molecular Biology

Article Title: miR-7704-Enriched Stem Cell-Derived Extracellular Vesicles Attenuate Hyperoxia-Induced Apoptosis and Oxidation in Lung Epithelial Cells

doi: 10.3390/cimb47110893

Figure Lengend Snippet: Western Blotting of Apoptosis Pathway after EV Therapy in BPD Model. ( A ) Cleaved-caspase 3 levels were significantly higher in the hyperoxia group compared to the normoxia group. After miR-7704-HUCMSCs-Exo treatment, cleaved-caspase 3 levels decreased significantly. No significant change in the level of cleaved-caspase 3 was observed after HUCMSC-Exo treatment. ( B ) Caspase 7 and FasL levels were significantly higher in the hyperoxia group compared to the normoxia group, while Bcl-2 levels were significantly lower. After miR-7704-HUCMSC-Exo treatment, cleaved-caspase 7 and FasL levels decreased significantly, while Bcl-2 levels increased significantly. No significant changes in FasL and Bcl-2 levels were observed after HUCMSC-Exo treatment. ( C ) No significant changes were observed in caspase 8 and caspase 9 levels in A549 cells under hyperoxic conditions.

Article Snippet: After thawing in liquid nitrogen, human type II alveolar epithelial A549 cells (ATCC CCL-185, Manassas, VA, USA) were cultured in F-12K Nutrient Mixture (Gibco, Waltham, MA, USA) containing 10% FBS and 1% penicillin/streptomycin for three passages.

Techniques: Western Blot