Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: SARS-CoV-2 infection of A549-hACE2 cells. A549-hACE2 cells were infected with SARS-CoV-2 (strain USA-WA1/2020) at an MOI of 1 for 24 h. (A) Immunofluorescent staining was performed to visualize infected cells by the presence of SARS-CoV-2 nucleocapsid (green). Nuclei of cells are stained with DAPI (blue). (B) SARS-CoV-2 spike RNA in infected cells ( n = 3, biological triplicate) was quantified by RT-qPCR. ND, not detected.

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Infection, Staining, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: Epitranscriptomic m 6 A microarray of SARS-CoV-2-infected A549-hACE2 cells. (A) Schematic overview of the method. Total cellular RNA from each sample (SARS-CoV-2-infected and mock-infected controls, biological triplicate, n = 3 each group) was used for immunoprecipitation using an m 6 A-specific antibody. Methylated and unmethylated RNA fractions were fluorescently labeled (Cy3 or Cy5) prior to array hybridization (refer to Materials and Methods for details). (B) Volcano plot of transcripts containing higher (red) and lower (blue) levels of m 6 A modification in infected cells compared to mock-infected control cells. The miRNA precursor (pre-mir-4486) with the most significant m 6 A change is labeled.

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Microarray, Infection, Immunoprecipitation, Methylation, Labeling, Hybridization, Modification, Control

Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: Summary of differentially modified transcripts by type in SARS-CoV-2-infected A549-hACE2 cells versus mock-infected control cells ^a

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Modification, Control

Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: Selected differentially m 6 A-modified transcripts in SARS-CoV-2-infected A549-hACE2 cells versus mock-infected control cells by m 6 A quantity ^a

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Control

Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: Selected differentially m 6 A-modified transcripts in SARS-CoV-2-infected A549-hACE2 cells versus mock-infected control cells by percent modified ^a

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Control, Modification

Journal: Microbiology Spectrum

Article Title: Epitranscriptomic N 6 -Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells

doi: 10.1128/spectrum.03943-22

Figure Lengend Snippet: Validation of differential methylation for selected transcripts in A549-hACE2 cells. (A) Schematic of m 6 A RIP. A549-hACE2 cells were mock infected or infected with ΔS-VRP(G) for 24 h. Total RNA was used for m6A-RIP. (B) Total RNA from cells infected with ΔS-VRP(G) was used for m 6 A-RIP. The fold enrichment of m 6 A-modfied RNA in the IP fraction of infected cell RNA versus mock-infected controls is indicated. (C) Total cellular RNA from mock-infected or ΔS-VRP(G)-infected cells was used for RT-qPCR analysis of total levels of mature miR-4486. (D) Total cellular RNA from mock-infected or ΔS-VRP(G)-infected cells was used for m 6 A RNA-IP to determine the levels of m 6 A-modified mature miR-4486. (C and D) Statistical significance was determined by t test compared to mock control. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Article Snippet: A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma).

Techniques: Methylation, Infection, Quantitative RT-PCR, Modification, Control

Journal: Oncology Letters

Article Title: Antitumor activity of nimotuzumab in combination with cisplatin in lung cancer cell line A549 in vitro

doi: 10.3892/ol.2018.7923

Figure Lengend Snippet: Cyclin D1 expression in A549 cells in groups A-E following treatment for 48 h. Lanes: A, nimotuzumab; B, cisplatin; C, nimotuzumab followed by cisplatin; D, nimotuzumab and cisplatin simultaneously; and E, untreated control.

Article Snippet: Cell culture The A549 human lung adenocarcinoma epithelial cell line (supplied by the Central Laboratory of Qinhuangdao No. 1 People's Hospital) was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd., Huzhou, China), 1 U/ml penicillin and 1 mg/ml streptomycin at 37°C, with 5% CO 2 and 95% humidity.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: ZnO nanocrystals derived from organometallic approach: Delineating the role of organic ligand shell on physicochemical properties and nano-specific toxicity

doi: 10.1038/s41598-019-54509-z

Figure Lengend Snippet: MTT cell viability evaluation and observation of the morphological changes of A549 (cancer) and MRC-5 (normal) cells after 24 h of incubation with NCs: ( a ) ZnO-MAA, ( b ) ZnO-MEAA, ( c ) ZnO-MEEAA. Experimental data were expressed as the mean of cell viability ± standard deviation (SD) of at least four individual experiments with six replicate wells. Asterisks denote statistical significance at p < 0.05.

Article Snippet: A cancer human alveolar epithelial cells (A549) and human fetal lung fibroblast cells (MRC-5) were purchased from the American Type Culture Collection (ATCC).

Techniques: Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: ZnO nanocrystals derived from organometallic approach: Delineating the role of organic ligand shell on physicochemical properties and nano-specific toxicity

doi: 10.1038/s41598-019-54509-z

Figure Lengend Snippet: The effect of ZnO NCs on intracellular ROS production. The A549 and MRC-5 cells were treated with 5, 15 and 25 µg/mL ZnO NCs for 24 h prior to the ROS determination including addition of DCFH-DA for 30 min followed by fluorescence measurement. The values are represented as mean ± S.D. of three individual experiments.

Article Snippet: A cancer human alveolar epithelial cells (A549) and human fetal lung fibroblast cells (MRC-5) were purchased from the American Type Culture Collection (ATCC).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: ZnO nanocrystals derived from organometallic approach: Delineating the role of organic ligand shell on physicochemical properties and nano-specific toxicity

doi: 10.1038/s41598-019-54509-z

Figure Lengend Snippet: The apoptosis rate in A549 and MRC-5 cells treated with ZnO-MAA ( b ), ZnO-MEEAA ( c ) and untreated with ZnO-AAA NCs cells ( d ) detected using flow cytometry. The percentage of early and late apoptotic cells is presented in graph ( a ). Flow charts: lower right quadrant, Annexin V positive and PI negative cells indicates early apoptotic cells; upper right quadrant, Annexin V and PI-positive cells represents necrotic or late apoptotic cells.

Article Snippet: A cancer human alveolar epithelial cells (A549) and human fetal lung fibroblast cells (MRC-5) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Influenza A alters host miRNA expression. (A) PCR analysis of individual miRNAs that showed differential expression in microarray analysis. miRNA isolated after 3 h of exposure to influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), (MOI of 3). The fold change represents the change in miRNA expression in infected cells relative to that obtained in uninfected cells. (B) A549 cells were infected with 3MOIs of influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), for 3 h and matrix copy numbers were determined by RT-PCR. (C) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza. Data are expressed as ± standard error of the mean (SEM). n=4 (independent experiments) with triplicate replicates (*=p<0.05, **=p<0.01, ***=p<0.001). (D) PCR analysis of individual miRNAs in human bronchial epithelial cells (HBEpC) exposed to influenza A (A/WS/33 (H1N1), and A/Aichi/2/68 (H3N2)). miRNA isolated after 3 h of exposure to influenza A (MOI of 3). The fold change represents the change in miRNA expression in infected cells relative to that of uninfected cells. (E) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (F) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza A. p<0.05 was considered significant. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that of uninfected cells. Data are expressed as ± standard error of the mean (SEM). n=4 (independent experiments) with triplicate replicates (*=p<0.05, **=p<0.01, ***=p<0.001).

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Quantitative Proteomics, Microarray, Isolation, Infection, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: miRNA-548an and NS1ABP mRNA expressions in A549 cells infected with different MOIs of influenza A. Differentially expressed miRNAs identified in the microarray analysis were screened for a potential role in influenza propagation and miRNA-548an was selected for further analysis. A549 cells were infected for 3 h with increasing MOIs of influenza. (A) miRNA-548an expression in uninfected and infected A549 cells was normalized to expression of let-7 as a housekeeping miRNA. (B) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to increasing MOIs of influenza. p<0.01 was considered significant. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) A549 cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Data are expressed as ± SEM. **=p<0.01, ***=p<0.001. n=4 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Infection, Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Time course showing the expression levels of miRNA-548an and NS1ABP mRNA in cells infected with influenza A. (A) A549 cells were infected with 1 MOI of Influenza A and expression of miRNA-548an normalized to let-7 miRNA is shown over 0–6 h of infection. (B) A549 cells were infected with 1 MOI of Influenza A and expression of NS1ABP mRNA normalized to GAPDH mRNA is shown over 0–6 h of infection. The fold change represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (D) Protein expression by Western blot analysis in cells infected with influenza A for up to 3 h and α-tubulin was used as a loading control. Data are expressed as ± SEM. ***=p<0.001, **=p<0.01, *=p<0.05; n=3 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Down or up-regulation of miRNA 548an alters the expression of NS1ABP mRNA. (A) Uninfected A549 cells were transfected for 48 h with 50 nM or 100 nM of either miRNA 548an inhibitor or a scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (B) After 48 h, transfected cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. n=3 (independent experiments) with triplicate replicates. (C) A549 cells were transfected for 48 h with 12.5 nM, 25 nM, or 50 nM of miRNA-548an mimic or scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (D) A549 cells were transfected for 48 h with miRNA-548an mimic or scrambled oligonucleotide. After 48 h, cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (E) NS1ABP expression profile after 30 and 48 h of transfection with mimics in A549 cells. (F) NS1ABP expression profile after 30 and 48 h of transfection with inhibitor in A549 cells. The fold change represents the change in NS1ABP mRNA expression and influenza A Matrix copy number in cells transfected with the inhibitor relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **=p<0.01 and *=p<0.05. n=4 (independent experiments) with triplicate replicates.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Transfection, Control, Infection

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: NS1ABP protein expression is modulated by miRNA 548an. (A) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor or scrambled oligonucleotide. (B) Flow cytometry analysis of A549 cells transfected for 48 h with 25 nM miRNA-548an mimic or scrambled oligonucleotide. (C) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor and then infected with 1 MOI of influenza for 3 h. Expression of NS1ABP protein in cells transfected with the miRNA-548an inhibitor (green line), or scrambled oligonucleotide as control (black line) is shown. (D) Flow cytometry analysis in A549 cells transfected for 48 h with miRNA-548an mimic (green line) or scrambled oligonucleotide (black line) and then infected with 1 MOI of influenza for 3 h. The gray shaded or red shaded regions in the graph represent the isotype control. (E) Geometric mean fluorescent intensity (MFI) was measured from 3 independent experiments with triplicate replicates. ###=p<0.001whencomparing cells transfected with inhibitor to cells transfected with the scrambled oligonucleotide; ##=p<0.01when comparing cells transfected with mimic to cells transfected with the scrambled oligonucleotide **=p<0.01 when comparing cells transfected with mimic or inhibitor.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Flow Cytometry, Transfection, Infection, Control

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Visualization of NS1ABP protein expression following modulation of miRNA-548an. A549 cells were transfected for 48 h with either the scrambled oligonucleotide (negative control) (top panel), miRNA-548an mimic (middle panel), or miRNA-548an inhibitor (third panel), and uninfected cells (bottom panel). Transfected cells were infected 1 MOI of influenza A for 3 h and expression of NS1ABP protein (green) and influenza nucleoprotein (red) was determined by immunofluorescence. DAPI (blue) represents the stained nucleus of the cells (not shown separately).

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Expressing, Transfection, Negative Control, Infection, Immunofluorescence, Staining

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Apoptotic pattern of cells transfected with miRNA-548an mimic or inhibitor and infected with influenza A. (A and C) A549 cells were transfected for 48 h with the scrambled oligonucleotide or (B) miRNA-548an mimic, or (D) miRNA-548an inhibitor. Transfected cells were then infected with 1 MOI of influenza A for 3 h. The cells were immune-fluorescently labeled with annexin V and apoptotic cells were analyzed by flow cytometry. Transfection did not change the proportion of necrotic cells detected by propidium iodide (PI) staining. Graph is a representative plot from 3 independent experiments and data presented as percentage of cells in each quadrant. Left upper quadrant=necrotic cells; left lower quadrant=viable cells; right lower quadrant=apoptotic cells; right upper quadrant=late apoptotic cells in necrotic state.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Transfection, Infection, Labeling, Flow Cytometry, Staining

Journal: Virology

Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

doi: 10.1016/j.virol.2013.08.031

Figure Lengend Snippet: Effect of miRNA 548an mimic on viral replication. (A) A549 cells were transfected for 48 h with 25 nM miRNA-548an mimic or a scrambled oligonucleotide. After 48 h, the cells were infected with increasing MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Expression of influenza Matrix gene copies in cells transfected with the mimic is expressed relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **=p<0.01, and *=p<0.05. n=3 (independent experiments) performed in triplicate.

Article Snippet: Cell culture Human alveolar epithelial A549 cells (CCL-34), American Type Culture Collection [ATCC, Manassas, VA] were cultured in standard F12K medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate.

Techniques: Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing