Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: Lipid secretion in A549 cells during long-term cultivation. A549 cells were cultured for 4, 7, 10, 15, and 25 days in low-glucose DMEM or low-glucose Ham’s F-12 medium. ( A ) Total cellular protein per dish as an indicator of culture expansion (cell accumulation). ( B ) Secretion of total lipids into the culture medium, assessed using [ 14 C]-acetate labeling. ( C ) Secretion of phospholipids and sphingolipids into the medium, quantified based on inorganic phosphate content (Pi). ( D ) Secretion of phosphatidylcholine (PC) into the medium, measured by mass spectrometry. ( A – D ) Data represent mean ± SD from at least three independent experiments (individual points shown). Values were normalized to mL of medium and mg of cellular protein, and additionally expressed relative to 4-day cultures in DMEM (left panels) or to 4-day cultures in the respective medium over time (right panels). Statistically significant differences between media (DMEM vs. Ham’s F-12; left panels) or between prolonged cultures and 4-day cultures (right panels) are indicated: * p < 0.05; ** p < 0.01; *** p < 0.001. RI, radioactivity.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Cell Culture, Labeling, Mass Spectrometry, Radioactivity

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: Intracellular phospholipid composition in A549 cells. A549 cells were labeled with [ 14 C]-acetate, and lipids extracted from cell homogenates were separated. ( A – D ) Relative amounts of individual phospholipids were calculated based on the radioactivity of individual spots. ( E ) Total radioactivity in phospholipids and ( F ) in total cell lipids, normalized to protein content. ( A – F ) Data represent mean ± SD from at least three independent experiments (individual points shown). Statistically significant differences between long-term cultures and 4-day cultures (asterisks) or between cells cultured in Ham’s F-12 and DMEM (hashtag) are indicated: * or # p < 0.05; ** or ## p < 0.01; *** or ### p < 0.001. CL, cardiolipin; DPM, disintegrations per minute; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipids; PS, phosphatidylserine; RI, radioactivity.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Labeling, Radioactivity, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: PG biosynthesis and secretion in A549 cells. ( A ) Relative cellular PG levels, quantified from the radioactivity of TLC-separated lipid spots. ( B ) PG secretion into the culture medium, determined by mass spectrometry. ( C ) PGS1 enzyme activity measured in cell homogenates cultured in DMEM. ( D ) Representative TLC image showing radioactive products of the PGS1 assay. Data represent mean ± SD from at least three independent experiments (individual data points shown). Statistically significant differences between long-term cultures and 4-day cultures are indicated by asterisks, and between cells cultured in Ham’s F-12 and DMEM by hashtags are indicated: * p < 0.05; ** or ## p < 0.01; *** or ### p < 0.001. PG, phosphatidylglycerol; PL, total phospholipids.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Radioactivity, Mass Spectrometry, Activity Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: Mitochondrial respiration and glycolytic activity in A549 cells. A549 cells were cultured in DMEM throughout the experiment. ( A ) Basal oxygen consumption rate was measured in intact cells as an indicator of mitochondrial respiration. ( B ) Extracellular acidification rate (ECAR) was determined as an indicator of glycolytic activity. ( C ) Representative immunoblot showing protein levels of mitochondrial respiratory chain subunits SDHB (Complex II, CII), UQCRC2 (Complex III, CIII), COXII (Complex IV, CIV), and ATP5A (ATP synthase, CV) (left), with No-Stain™ total-protein labeling shown for normalization (right). ( D – G ) Quantification of individual respiratory complex subunits and ATP synthase levels during the cultivation period, normalized to No-Stain™ total protein per lane. Data represent mean ± SD from at least three independent experiments (individual data points shown). Statistically significant differences between long-term cultures and 4-day cultures are indicated: * p < 0.05; *** p < 0.001.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Activity Assay, Cell Culture, Western Blot, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: Phenotypic markers and lipid features of A549 cells. A549 cells were cultured in DMEM throughout the experiment. ( A ) Expression of proliferation markers KI67 and PCNA determined by RT-qPCR. ( B ) Expression of phenotypic markers CAV1 , PDPN , AGER , and CDKN1A determined by RT-qPCR. ( C ) Expression of the ATII marker ABCA3 determined by RT-qPCR. ( D ) Intracellular SP-A protein levels during 25 days of long-term static cultivation (left) and representative immunoblot analysis of SP-A and β-actin (right), used for normalization. ( E ) Cellular cholesterol and neutral lipid levels (TAG and SE) determined spectrophotometrically at 475 nm after sulfuric acid staining. ( A – E ) Data represent mean ± SD from at least two independent experiments (individual points shown) and were normalized to 4-day cultures. Statistically significant differences between long-term cultures and 4-day cultures are indicated: * p < 0.05; ** p < 0.01; *** p < 0.001. SE, sterol esters; SP-A, surfactant protein A; TAG, triacylglycerols.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Marker, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Long-Term Static Cultivation Alters Lipid Metabolism and Bioenergetic Capacity in A549 Cells

doi: 10.3390/ijms27083417

Figure Lengend Snippet: Inflammasome-associated transcripts and loss of viability in long-term cultured A549 cells. A549 cells were cultured in DMEM throughout the experiment. ( A ) Cell viability assessed by flow cytometry using Annexin V (AV) and propidium iodide (PI) staining. Data represent mean ± SD from three independent experiments. ( B ) Representative flow cytometry dot plots. ( C ) Relative transcript levels of C3 , CASP1 , and IL1B determined by RT-qPCR. ( D ) Intracellular Ca 2+ concentration. ( E ) Reactive oxygen species (ROS) fluorescence intensity. ( F ) Relative level of cleaved gasdermin D-N (GSDMD-N). ( G ) Representative immunoblot showing full-length GSDMD and cleaved GSDMD-N (left), with No-Stain™ total-protein labeling used for normalization (right). ( C – F ) Data represent mean values from at least three independent experiments (individual points shown), normalized to day 4 cultures. Statistically significant differences between long-term cultures and 4-day cultures are indicated: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A549 human alveolar basal epithelial cells (CCL-185, ATCC, Manassas, VA, USA), originally derived from a human lung adenocarcinoma [ ], were cultured in either low-glucose Ham’s F12 (N3520, Sigma-Aldrich, St. Louis, MO, USA) or low-glucose DMEM (LM-D1099, Biosera, Nuaille, France), supplemented with 10% fetal bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin/streptomycin (XC-A4122, Biosera, Nuaille, France).

Techniques: Cell Culture, Flow Cytometry, Staining, Quantitative RT-PCR, Concentration Assay, Fluorescence, Western Blot, Labeling