Journal: Journal of the Chinese Medical Association : JCMA

Article Title: TRIM72 mediates lung epithelial cell death upon hyperoxia exposure

doi: 10.1097/JCMA.0000000000000413

Figure Lengend Snippet: Immunohistochemistry results (A) and Western blotting results and quantitative data (B, C) for TRIM72 expression in 7-day-old and 14-day-old rats in the normoxia and O 2 -enriched groups. TRIM72 immunoreactivity was observed in endothelial cells (black arrow), superior surface of bronchial epithelial cells (red arrow), and alveolar type I (short black arrow) and II cells (short red arrow). The normoxia group showed faint immunoreactivity in bronchial epithelial cells (red arrow) and alveolar type I (short black arrow) and II cells (short red arrow). The rats in the O 2 -enriched group exhibited significantly higher TRIM72 expression than those in the normoxia group on postnatal days 7 and 14. * p < 0.05.

Article Snippet: RLE-6TN, a rat alveolar type II epithelial cell line, and A549, a human lung carcinoma epithelial cell line (ATCC, Manassas, VA, USA), were maintained in an F-12 medium in 75-cm 2 tissue culture flasks at 37.8°C in 5% CO 2 and 95% air.

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: Characterization of A549 and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Expressing, Western Blot, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII induce cytotoxicity in A549 and A549/DDP cells. (A) Chemical structure of PPI and PPVII. (B) A549 and A549/DDP cells were treated with different dose of PPI (0.5–8 µg/ml) and PPVII (0.5–8 µg/ml) for 24 h, respectively. In A549 cells, the cell inhibition rate of 0.5 µg/ml PPI was significantly different from other concentration groups (**P<0.01); 1.0 µg/ml was significantly different from 4.0 and 8.0 µg/ml (**P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml (**P<0.01). As for A549/DDP cells, the cell inhibition rates of 0.5 µg/ml PPI and 1.0 µg/ml were both significantly different from other concentration groups ( ## P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml ( ## P<0.01). In A549 cells, there were significant differences in cell inhibition rates of PPVII among different concentration groups (**P<0.01); as for A549/DDP cells, there were significant differences among different concentration groups except for 4.0 and 8.0 µg/ml ( ## P<0.01). PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Inhibition, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII possess chemo-sensitizing effects on A549/DDP cells. (A) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPI or 0.75 µg/ml PPI, respectively for 24 h. (B) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPVII or 0.75 µg/ml PPVII, respectively for 24 h, cell viability was measured with Cell Counting Kit-8 assay. Data were presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. DDP treated group. # P<0.05 and ## P<0.01. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reduce the IC 50 value of cisplatin in A549/DDP cells

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques:

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. Apoptosis of cells was detected by Annexin V-FITC/PI staining and flow cytometry. The apoptotic percentage is shown as a bar graph. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis through the P53 pathway and caspases-dependent pathway. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. The protein expression levels of P53, Bax, Bcl-2 (A), PARP, C-PARP, pro-Caspase-3, C-Caspase-3 (B) were measured using western blotting. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; C-PARP, cleaved-poly (ADP-ribose) polymerase 1.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reverse EMT and suppress the CIP2A/AKT/mTOR pathway. A549/DDP cells were incubated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, then cells were harvested for further western blotting analysis. (A) The protein expression levels of E-cadherin, vimentin, α-SMA in A549/DDP cells were measured. (B) The protein expression levels of CIP2A, p-AKT, AKT, p-mTOR, m-TOR were measured. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; p-AKT, phosphorylated-protein kinase B; mTOR, mammalian target of rapamycin; CIP2A, cancerous inhibitor of protein phosphatase 2A; SMA, smooth muscle actin; E, epithelial.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Incubation, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: Effects of PUFAs on binding of RBD sequence of SARS-CoV-2 to human ACE2 receptor. ( A ) Binding of RBD sequence of SARS-CoV-2 spike protein to immobilized hACE2 receptor. HRP-conjugated RBD sequence was treated with indicated FAs at different concentrations for 30 min. followed by incubation for 15 min. with hACE2 receptors immobilized on plate. HRP signal was measured at 450 nm. ( B) Binding of A549 cells expressing SARS-CoV-2 eGFP-spike protein in the present of selected FAs at different concentrations to soluble hACE2 receptor. Binding was performed on plates with immobilized human monoclonal antibody against hACE2 receptor at 10 µg/ml concentration and detected as green fluorescence signal. Data are presented as % of control ± SD; # p ≤ 0.05, ∆ p ≤ 0.01, * p ≤ 0.001. Control—0.025% DMSO, positive and negative controls were provided by the manufacturer.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Sequencing, Incubation, Expressing, Concentration Assay, Fluorescence, Control

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: Effects of linolenic acid and EPA on viability of ACE2 expressing A549 cells and on human ACE2 receptor. ( A ) Viability of A549/hACE2 cells after 1 h, 3 h, and 48 h incubation with linolenic acid and EPA at different concentrations using MTT assay. Cell viability is expressed as change in absorbance at 570 nm in % compared to lipid-free control ± SD; positive control—100% dead cells, negative control—addition-free sample. ( B ) Binding of linolenic acid and EPA at indicated concentrations to hACE2 receptor (1.0 μg/ml) immobilized on the plate using human primary anti-ACE2 antibody at 1:500 dilution and HRP-conjugated secondary antibody at 1:1000 dilution, and measuring chemiluminescence signal. Data are presented as % of lipid-free control ± SD; control—0.025% DMSO, positive control—50% DMSO. ( C) Activity of hACE2 upon treatment with selected FAs and indicated concentrations. Purified hACE2 enzyme at 0.1 ng/µl was incubated with linoleic acid and EPA at different concentrations for 1 h at RT followed by addition of 25 µl fluorogenic substrate for 30 min. Fluorescence signal was measured at Ex/Em = 535/595 nm using spectrofluorimeter. Data are presented as % of lipid-free control ± SD; * p ≤ 0.001. Control—0.025% DMSO, positive control—10% DMSO.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Expressing, Incubation, MTT Assay, Control, Positive Control, Negative Control, Binding Assay, Activity Assay, Purification, Fluorescence

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: Effects of linolenic acid and EPA on SARS-CoV-2 pseudo-virion binding to human ACE2 receptor. Binding SARS-CoV-2 spike protein encapsulated pseudo-virions to A549 cells stably overexpressing human ACE2 receptor was evaluated with monoclonal antibody. ( A ) Spike-pseudo-virions were treated with indicated FAs at different concentrations for 1 h before inoculation, added simultaneously or 1 h after inoculation of the virions into hACE2/A549 cells. Next, wells were incubated for 1 h at 37 O C, washed and binding was evaluated by adding primary anti-spike protein monoclonal antibody at 1:1000 dilution followed by secondary HRP-conjugated antibody at 1:2500 dilution and signal measurement at 450 nm. ( B ) Spike-pseudo-virions were treated with indicated FAs at different concentrations for 1 h before inoculation, added simultaneously or 1 h after inoculation of the virions into hACE2/A549 cells. Next, wells were incubated for 3 h at 37 O C, washed and binding was evaluated by adding primary anti-spike protein monoclonal antibody at 1:1000 dilution followed by secondary HRP-conjugated antibody at 1:2500 dilution and signal measurement at 450 nm. Data are presented as % of control ± SD; # p ≤ 0.05, ∆ p ≤ 0.01, * p ≤ 0.001. Controls—0.025% DMSO, positive and negative controls were provided by the manufacturer.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Stable Transfection, Incubation, Control

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: SARS-CoV-2 eGFP-luciferase-pseudo-virion binding and cellular entry. Binding and entry of SARS-CoV-2 pseudo-virions with encapsulated eGFP-luciferase spike protein was evaluated without spinfaction and with spinfaction. ( A ) A549 cells stably overexpressing hACE2 receptor were inoculated (no spinfection) with SARS-CoV-2 pseudo-virions treated with indicated FAs at different concentrations and time. After 48 h post-inoculation time, transduction efficacy was measured according to luciferase activity. ( B ) Binding and entry of SARS-CoV-2 pseudo-virions with encapsulated eGFP-luciferase Spike protein. A549 cells stably overexpressing hACE2 receptor were inoculated (spinfection) with SARS-CoV-2 pseudo-virions treated with indicated FAs at different concentrations and time. After 48 h post-inoculation time, transduction efficacy was measured according to luciferase activity. Data are presented as % of control ± SD; # p ≤ 0.05, ∆ p ≤ 0.01, * p ≤ 0.001. Control—0.025% DMSO, positive control—bald SARS-CoV-2 eGFP-luciferase-pseudo-virions, negative control—ΔG-luciferase rVSV pseudo-typed particles; red fame—concentrations that showed 85–100% cytotoxicity.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Luciferase, Binding Assay, Stable Transfection, Transduction, Activity Assay, Control, Positive Control, Negative Control

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: Effect of selected FAs on fusion to human ACE2 receptor overexpressing A549 cells. ( A ). Cell–cell fusion of A549 cells expressing eGFP spike protein with A549 cells stably expressing human ACE2 receptor. A549 cells expressing eGFP spike protein were pre-treated with indicated FAs at different concentrations for 1 h at 37 °C and co-cultured for additional 4 h at 37 °C with A549 cells stable expressing human ACE2 receptor. The scale bar indicates 250 µm. ( B ) Quantitative analysis of formed syncytia. Experiments were done in triplicate and repeated three times. Data are presented as % of control ± SD; * p ≤ 0.001. Control—0.025% DMSO, positive control—20 μg/ml anti-ACE2 antibody.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Expressing, Stable Transfection, Cell Culture, Control, Positive Control

Journal: Scientific Reports

Article Title: Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

doi: 10.1038/s41598-021-84850-1

Figure Lengend Snippet: Effect of selected FAs on TMPRSS2 and Cathepsin L proteases. ( A ) Purified TMPRSS2 enzyme at 1 µM was incubated with selected FAs at different concentrations for 1 h at RT followed by addition of fluorogenic substrate at 10 µM concentration for 30 min. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (upper panel). A549 cells were treated with indicated FAs at different concentrations for 3 h and 48 h at 37 °C and enzymatic activity was measured by addition of 0.2 mM fluorogenic substrate and incubation for 30 min. at 37 °C. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (lower panel). Data are presented as % of control ± SD; # p ≤ 0.05, ∆ p ≤ 0.01, * p ≤ 0.001. Control—0.025% DMSO, positive control—50–100 μM camostat mesylate. ( B ) Purified cathepsin L enzyme at 0.02 ng/µl was incubated with selected FAs at different concentrations for 1 h at RT followed by addition of fluorogenic substrate at 10 µM concentration for 30 min. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (upper panel). A549 cells were treated with indicated lipids at different concentrations for 24 h at 37 °C and enzymatic activity was measured by addition of 0.2 mM fluorogenic substrate and incubation for 30 min. at 37 °C. Fluorescence signal was measured at Ex/Em = 360/535 nm using spectrofluorimeter (lower panel). Data are presented as % of control ± SD; # p ≤ 0.05, * p ≤ 0.001. Control—0.025% DMSO, positive control—0.1 μM E-64. ( C ) Western blot analysis of TMPRSS2 and cathepsin L expression in A549 cells treated with indicated FAs with different concentration for 48 h. Detection was done using rabbit anti-TMPRSS2 monoclonal antibody at 1:1000 and mouse anti-cathepsin L antibody at 1:200. Experiments were done in triplicate and repeated three times. Data are presented as % of lipid-free control ± SD.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor, was obtained from GenScript (Piscataway, NJ).

Techniques: Purification, Incubation, Concentration Assay, Fluorescence, Activity Assay, Control, Positive Control, Western Blot, Expressing