Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: XPA is phosphorylated in cells on UV irradiation. A, A549 cells were treated with 20 J/m2 UV or mock treated. Total cell lysates were harvested at 4 hours after UV irradiation and then treated with 200 units of CIAP (Promega) for 1 hour at 37°c in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of 50 mmol/L glycerophosphate (G.P.) or mock treated (lanes 1 and 2). The treated cell lysates were then subjected to Western blotting and probed with anti-XPA (a) and anti-RPA32 (b), respectively. c, cells were treated with 20 J/m2 UV or mock treated and total cell lysates were harvested for immunoprecipitation assays with anti-XPA antibody. The immunoprecipitated XPA was treated with CIAP or mock treated and then analyzed by Western blotting with anti-XPA antibody. B, A549 cells were grown overnight in phosphate-depleted medium before irradiation with 20 J/m2 UV. Then, 32P-labeled orthophosphoric acid was added and cells were further incubated for 8 hours before harvest. Immunoprecipitation assay was done with anti-XPA antibody. Immunoprecipitates were separated on SDS-PAGE and radiolabeled proteins were detected (left). Immunoprecipitated endogenous XPA was probed by Western blotting (right). C, cells were UV irradiated or mock treated and then cytoplasmic and nuclear extractions were separated on SDS-PAGE for Western blot analysis using anti-XPA antibody. D, a, cells were irradiated with 20 J/m2 of UV and the cytoplasmic fraction (lane 1) was isolated. The nuclear pellet was then sequentially extracted with buffer of increasing salt concentration (lanes 2-5). NM, nuclear matrix (lane 6). b, nuclear extracts were prepared at indicated times after 20 J/m2 UV treatment of cells. c, cells were irradiated with indicated doses of UV and the nuclear extracts were prepared at 4 hours after UV treatment.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Irradiation, Western Blot, Immunoprecipitation, Labeling, Incubation, SDS Page, Isolation, Concentration Assay

Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: ATR is the major kinase responsible for cellular XPA phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or HeLa cells were transfected with indicated siRNA and then treated with 20 J/m2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Phospho-proteomics, Irradiation, Incubation, Western Blot, Transfection

Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: ATR interaction and colocalization with XPA in cells after UV irradiation. A, total cell lysates prepared from UV-treated or mock-treated A549 cells were used for coimmunoprecipitation assays with anti-ATR antibody. Proteins from the immunoprecipitates were detected by Western blotting using anti-XPA and anti-ATR antibodies. As controls, 10% of the total volumes of the whole cellular lysates used for the coimmunoprecipitation were also included. B, top, total cell lysates prepared from A549 cells were used for coimmunoprecipitation assays with anti-XPA antibody; bottom, whole-cell extracts prepared from 2 × 106 cells were mixed with 2 μg of His-XPA and incubated at 4°c for 10 to 14 hours. The XPA-bound proteins were probed by anti-ATR antibody. C, total cellular lysates were incubated with anti-ATR antibodies for 4 to 6 hours, followed by 1-hour incubation with protein A/G-agarose beads. The immunoprecipitates were washed thrice with PBS containing 0.5% NP40 and further incubated with the buffer of high concentration of salt [15 mmol/L Tris-Cl (pH 7.5), 0.6 mol/L NaCl, 0.1% NP40] for 30 minutes at 4°c. After centrifugation and washing, purified His-XPA was added and further incubated in 500 AL of XPA binding buffer [40 mmol/L HEPES-KOH (pH 7.5), 75 mmol/L KCl, 8 mmol/L MgCl2, 1 mmol/L DTT, 5% glycerol and 100 Ag/mL bovine serum albumin, 0.1% NP40] for 4 to 6 hours. The bound proteins were detected by Western blotting with anti-XPA antibody. LC, loading control (20 ng purified His-XPA). D, cells were treated with 20 J/m2 UV or mock treated, followed by 4-hour incubation. After extraction of cytoplasmic proteins with PBS containing 0.5% NP40, cells were fixed and incubated with anti-XPA and anti-ATR antibodies. Cells were then stained with fluorescent dye-linked secondary antibodies and visualized by fluorescence microscopy. b and f, red, anti-ATR stained cells; c and g, green, anti-XPA stained cells; d and h, merged images of the anti-XPA and anti-ATR stained cells; a and e, 4‘,6-diamidino-2-phenylindole-stained nuclei.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Irradiation, Western Blot, Incubation, Concentration Assay, Centrifugation, Purification, Binding Assay, Control, Extraction, Staining, Fluorescence, Microscopy

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Effect of JNK inhibition on A549 cell viability as a function of cisplatin concentration. (a, b) 7.5 μg/mL concentration of cisplatin becomes lethal in combination with JNK inhibitor SP600125. However, at higher cisplatin concentrations, the inhibitor either protects against cisplatin-induced cell death (a) or does not have any effect (b). (c) Similar effects are observed in the colon cancer cell line DLD-1. (d–g) SP600125 potentiates the appearance of cells with apoptotic morphology in 7.5 μg/mL cisplatin-treated A549 cells (d, e), in contrast to 30 μg/mL cisplatin-treated A549 cells (f, g). Relative cell viability is cell viability after 72 h treatment normalized by initial viability (measured by the MTT method). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. Fluorescent pictures were obtained with a mixture of AO/EB dyes as described in the section. SP—JNK inhibitor SP600125 (20 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Inhibition, Concentration Assay

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: JNK inhibitor SP600125 reduces JNK target transcription factor c-Jun phosphorylation in cisplatin-treated A549 cells. Different concentrations of cisplatin were used. (a) Representative Western blots from 20 and 40 h of treatments are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. (b) Repeated addition (after 24 h) of SP potentiates suppression of c-Jun phosphorylation in response to cisplatin treatment. (c) Repeated addition of SP strengthens the protective effect of JNK inhibition at 15−30 μg/mL concentrations of cisplatin in A549 cells. SP—JNK inhibitor SP600125 (20 μM). p *** < 0.0005, N = 3.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Western Blot, Staining, Inhibition

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Cisplatin concentration-dependent effect of different JNK inhibitors on A549 cell viability. Different JNK inhibitors show the same dependence on cisplatin concentration on cell viability. Statistically significant reduction in cell viability is observed at 7.5 μg/mL cisplatin in combination with 10 μM AS601245 (a), 10 μM bentamapimod (b), 2 μM JNK inhibitor IX (c), and 5 μM XG-102 (d). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. (e) Reduction in c-Jun phosphorylation upon the addition of JNK inhibitor XG-102, as determined by Western blot. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Activation of TP53 in A549 cells in response to different concentrations of cisplatin. (a, b) Expression and phosphorylation at serine-6 is induced by cisplatin and is maximal at 15 μg/mL (6 h of cisplatin treatment). (c, d) Prolonged and increasing expression and phosphorylation of TP53 in cells treated with either 7.5 μg/mL or 30 μg/mL of cisplatin. (e) TP53 activator nutlin-3a potentiates cisplatin-induced decrease in cell viability. (f) Nutlin-3a reduces viability of cisplatin + SP600125-treated cells. C—control without cisplatin; Nut—nutlin-3a (10 μM); SP—SP600125 (20 μM). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented, p ** < 0.005, p *** < 0.0005, N = 4. (g) Nutlin induces expression of TP53. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Control, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Involvement of AKT signaling pathway in cisplatin-induced A549 cell death. (a) AKT protein level does not depend on cisplatin concentration. 6-h-long exposure is presented in the Western blot picture. (b) Phosphorylation/activation of AKT is cisplatin concentration-dependent. 6-h-long exposure is presented in the Western blot picture. (c) AKT protein level does not change during 40 h of cisplatin treatment. (d) Dynamics of AKT activation following cisplatin (7.5 or 30 μg/mL) treatment. (e, f) AKT inhibitor capivasertib enhances cell death at all concentrations of cisplatin used both in the absence (e) and presence (f) of JNK inhibitor SP600125. (g, h) GSK3-β inhibitor SB415286 protects A549 cells from cisplatin both in the absence (g) and presence (h) of JNK inhibition. Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. p*<0.05, p**<0.005, p***<0.0005, N = 4. (i) Capivasertib inhibits AKT activity as shown by the inhibition of AKT molecular target GSK3-β phosphorylation at serine-9. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. C—control without cisplatin; CAP—capivasertib (10 μM); SP—SP600125 (20 μM), SB—GSK3 inhibitor SB415286 (15 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Western Blot, Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Staining, Control

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Opposite changes in TP53 and AKT phosphorylation following SP600125 treatment of A549 cells exposed to different concentrations of cisplatin. (a) Expression of TP53 is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (b) Phosphorylation of TP53 (serine-6) is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (c) Phosphorylation of AKT (threonine-308) is decreased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as a loading control. SP—JNK inhibitor SP600125 (20 μM); 20 h of treatment.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Expressing, Western Blot, Staining, Control

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 1 Elevated miR-762 expression is associated with gefitinib resistance in NSCLC cells. a RT-qPCR analysis of miR-762 expression in different NSCLC cells. Relative expression levels of miR-762 were obtained in each sample by normalization of the expressions of miR-762 to that of the U6 snRNA signal. For presentation of data, expression levels of miR-762 from NuLi-1 cells were taken as 100% and the others were normalized accordingly. Each value is a mean ± S.E.M. from three independent experiments. Different superscript letters denote groups that are statistically different (P < 0.05). b The IC50 value (inhibitory concentration to produce 50% cell death) following a 48-h exposure to gefitinib was determined in PC-9 and PC-9/GR cells using MTT assay. c The IC50 value following a 48-h exposure to gefitinib was determined in A549 and A549/GR cells using MTT assay. d-e Characterization of miR-762 expression in different NSCLC cells using RT-qPCR. The value indicates the relative expression levels of miR-762 in the cells (PC-9/PC-9/GR and A549/A549/GR cells) at different batches of gefitinib resistant induction

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, MTT Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 2 IL-6 serves as a potential upstream regulator of miR-762 induction in NSCLC cells. a-b Characterization of expression levels of different cytokines in different NSCLC cells using RT-qPCR. Each value is a mean ± S.E.M. from three independent experiments. c A549 cells were incubated with different cytokines, including IL-1α (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) and IL-8 (50 ng/ml) for 24 h, followed by RT-qPCR analysis of miR-762 expression. d A549 cells were stimulated with different doses of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression. e A549 cells were stimulated with 10 ng/ml of IL-6 for different durations as indicated, followed by RT-qPCR analysis of miR-762 expression. f A549 cells were stimulated with 10 ng/ml of IL-6, in the presence or absence or co-treatment with 5 μM of WP1066, for 24 h, followed by RT-qPCR analysis of miR-762 expression. Inhibition of STAT3 activation was verified using immunoblotting analysis of pSTAT3 expression (upper panel). g A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA. 48 h later, knockdown of STAT3 in A549 cells was validated using immunoblotting. h 48 h after siRNA treatment, A549 cells were stimulated with 10 ng/ml of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Incubation, Inhibition, Activation Assay, Western Blot, Transfection, Knockdown

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 3 miR-762 upregulation desensitizes NSCLC cells to gefitinib treatment. a 48 h after transfection with miR-762 inhibitors or negative controls (NC), PC-9/GR and A549/GR cells were subjected to RT-qPCR analysis of miR-762 expression. b PC-9/GR and A549/GR cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). c Cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). d In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and ** P < 0.01 when comparing Inhibitors + vehicle to Inhibitors + gefitinib). e 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9 and A549 cells were subjected to RT-qPCR analysis of miR-762 expression. f PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). g PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). h In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and **P < 0.01 when comparing Mimics + vehicle to Mimics + gefitinib)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 4 ABR serves as the direct target of miR-762 in NSCLC cells. a Prediction of putative target genes of miR-762 by Target scan and miRDB programs. b PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by immunoblotting analysis of ABR expression. c PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by RT-qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). d PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. e PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by RT- qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). f Predicted miR-762 binding sites in the 3′-UTR of ABR gene. g miR-762 mimics/Mimics- NC (25 pmol/well) and pGL3-ABR 3’UTR-Luc reporter (0.25 μg/well), together with 0.001 μg of the Renilla luciferase reporter (Promega, Beijing, China), were co-transfected into subconfluent proliferating NIH/3 T3 cells for 24 h, followed by measurement of the relative luciferase activity (*P < 0.05)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 5 ABR overexpression alleviates miR-762-induced gefitinib resistance. a PC-9 and A549 cells that stably expressed the exogenous ABR was established as described in Materials and methods. PC-9/ABR and A549/ABR cells were transiently transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. b 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm. c 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell apoptosis was assayed using an ApoStrand™ ELISA Apoptosis Detection Kit at 405 nm. Different superscript letters denote groups that are statistically different (P < 0.05). d 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were subjected to a xenograft model to measure the in vivo gefitinib sensitivity, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 when comparing Mimics + gefitinib + vector to Mimics + gefitinib + pCMV3-ABR)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Over Expression, Stable Transfection, Transfection, Western Blot, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Western blot showing the expression of E1B-55K target genes in A549 cells infected with either wildtype (HA-tagged E1B-55K-expressing) virus or a ΔE1B-55K mutant (lacking E1B-55K expression). Cells were infected with MOI 30 and analyzed after 24 hours. This representative replicate illustrates the steady-state levels of p53, Mre11 and CDKN1A during infection (antibodies are listed in ). (B) Principal component analysis of mock-, wildtype- and ΔE1B-55K-infected A549 cells after differential gene expression analysis. (C) Volcano plot comparing gene expression between wildtype- and ΔE1B-55K-infected A549 cells. Genes with an adjusted P -value < 0.1 and a log₂ fold change < -1 are shown in blue, while those with an adjusted P -value < 0.1 and a log₂ fold change > 1 are shown in red. Genes belonging to the p53 transcriptional network (WikiPathways database) are highlighted with green circles. (D) Heatmap displaying row Z-scores of p53 pathway genes that were downregulated in (C) and are marked in green. The grey, black, and purple boxes at the top of the panel represent mock, wildtype, and ΔE1B-55K infection, respectively. (E) Global pathway analysis of differentially expressed genes comparing wildtype- with ΔE1B-55K-infected A549 cells using gene set enrichment analysis. The normalized enrichment scores of significantly up- and downregulated pathways are shown, with pathways meeting FDR < 0.1 highlighted in light blue/red and those with FDR < 0.05 in dark blue/red. p53-associated pathways are written in green. Red bars indicate pathways enriched in upregulated genes, while blue bars indicate pathways enriched in downregulated genes.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Western Blot, Expressing, Infection, Virus, Mutagenesis, Gene Expression

Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Interferon-alpha induction scheme in A549 and H1299 cells. The time points post-infection at which DNA is extracted for viral DNA quantification are illustrated below the timeline bar. (B) Western blot from one of the sequenced replicates, displaying selected viral- and IFN-induced proteins (antibodies are listed in ). Proteins associated with the p53 pathway and the immune response are marked in green and pink, respectively. The upper seven proteins were detected on medical X-ray films and the lower five proteins were visualized via the ChemoStar Plus. Additional replicates are shown in . (C) Virus yield was determined 24 hpi with MOI 30 by anti-DBP (B6-8) immunofluorescence staining. Bar graphs represent the mean virus yield of three independent experiments, error bars indicate SD. Black and purple bars represent wildtype and ∆E1B-55K virus, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively. Statistical significance was determined using two-tailed t-test. ** P -value < 0.01, * P -value < 0.05, ns P -value > 0.05. (D) Normalized viral mRNA counts of selected early (upper row) and late (lower row) genes. *adjusted P -value < 0.1; ns adjusted P -value > 0.1. Light grey, black, and purple box plots represent mock, wildtype virus, and ΔE1B-55K virus conditions, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Infection, Western Blot, Virus, Immunofluorescence, Staining, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Principal component analysis of mock-, wildtype- and ΔE1B-55K-infected A549 (left) and H1299 (right) cells after differential gene expression analysis. Circles and triangles indicate BSA and IFN treatment, respectively. (B) Pathway analysis of downregulated genes comparing wildtype with ΔE1B-55K infection upon IFN treatment using GSEA, showing the normalized enrichment score for pathways with FDR < 0.1 (light blue) and FDR < 0.05 (dark blue). The upper and lower bar graphs represent pathways enriched in infected A549 and H1299 cells, respectively. Only downregulated pathways are displayed; upregulated pathways are shown in and . Immune response-associated pathways are highlighted in pink and p53-associated pathways in green. The dotted line separates the two cell lines, as labeled on the right. (C) Normalized viral mRNA counts of selected p53 and immune-system associated pathways from wildtype- and ΔE1B-55K-infected A549 (top) and H1299 (bottom) cells treated with IFN. ****adjusted P -value < 0.0001, ***adjusted P -value < 0.001, *adjusted P -value < 0.1, ns adjusted P -value > 0.1. Light grey, black, and purple box plots represent mock, wildtype virus, and ΔE1B-55K virus conditions, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively. (D) Volcano plot comparing gene expression between wildtype- and ΔE1B-55K-infected A549 (top) and H1299 (bottom) cells treated with IFN. Genes with an adjusted P -value < 0.1 and log 2 fold change < 0 are colored in blue, while genes with an adjusted P -value < 0.1 and log 2 fold change > 0 were colored in red. Genes that belong to the IFN-associated gene network are shown with pink dots.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Infection, Gene Expression, Labeling, Virus

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 1 Decreased PICT1 protein expression in ATII cells in emphysema patients. Lung tissue and ATII cells were obtained from control non-smoker (N) and smoker (S) organ donors and emphysema patients (E). Panel I: A—PICT1 mRNA levels in lung tissue by RT-PCR. B—Representative Western blot images of PICT1 expression in lung tissue. C—Quantification of protein expression normalized to β-actin is shown. Panel II: A—PICT1 mRNA levels in ATII cells by RT-PCR. B—Representative Western blot images of PICT1 expression in ATII cells. C—Quantification of protein expression. Panel III: A – PICT1 was immunoprecipitated in lung tissue, followed by mass spectrometry analysis. Representative PICT1 (A) and TRIM22 spectrum (B) are shown. C TRIM22 mRNA expression in ATII cells by RT-PCR. D ATII cells were stained in lung tissue sections using SP-C (magenta), PICT1 (red), and TRIM22 (green) antibodies and DAPI (blue) followed by analysis by immunofluorescence (scale bar—5 μm). PICT1 fluorescence intensity in the nucleus (E) and cytoplasm (F) was quantified. G The ratio of nuclear to cytoplasmic PICT1 fluorescence intensity. H Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization in ATII cells. Data are shown as means ± SEM (N = 3—14 lungs per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Mass Spectrometry, Staining, Immunofluorescence, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 3 Decreased MRE11 protein levels in ATII cells in a murine model of emphysema. Wild-type mice were exposed to cigarette smoke for 8 months, as described in the Methods section, to induce emphysema. A Hematoxylin and eosin staining in murine lung tissue (scale bar 50 μm). Minimum (B), maximum (C), and mean (D) alveolar diameters were measured in lung tissue sections. E Representative micro-CT of the murine lung (scale bar-100 μm). F The intersection surface was quantified using micro-CT images. G Pict1 and H Mre11 mRNA levels were evaluated in lung tissue by RT-PCR. I Representative Western blotting images of PICT1 and MRE11 expression in lung tissue. PICT1 (J) and MRE11 expression (K) are quantified. L ATII cells in lung tissue sections were identified using SP-C (green). PICT1 (magenta), and MRE11 (red) antibodies, and DAPI (blue) by immunofluorescence (scale bar—5 μm). Quantification of PICT1 (M) and MRE11 (N) fluorescence intensity in ATII cells is shown. O Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization. Data are shown as means ± SEM (N = 3 – 8 mice per group). p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Staining, Micro-CT, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 6 Mitochondrial dysfunction in human primary ATII cells, A549 cells, and MLE15 cells. A PICT1 (red), TOM20 (green), and DAPI (blue) staining in lung tissue sections obtained from non-smokers (N), smokers (S), and emphysema patients (E) by immunofluorescence (scale bar—5 μm). ATII cells were identified using SP-C (magenta). B Pearson’s correlation coefficient for PICT1 and TOM20 co-localization in ATII cells is shown (N = 3 lungs per group). C Quantification of mitochondrial networks in ATII cells. D Mitochondrial respiration analysis in wild-type A549 cells and cells with PICT1 deletion treated with 20% cigarette smoke extract (CSE) for 24 h. Quantification of basal respiration (E) and maximum respiration (F) in A549 cells. G ATP-linked respiration after exposure to CSE relative to controls in A549 cells. QPCR was used to determine mtDNA amount (H), mtDNA damage (I), and common deletions (CD, J) in A549 cells. Representative histograms using MitoSOX staining and flow cytometry analysis (K) and the quantification of fluorescence intensity (L) in A549 cells. M Representative Western blotting images of MLE15 cells treated with NT (non-target) or PICT1 siRNA. N Histograms of MitoSOX staining by flow cytometry analysis (N) and quantification (O) in MLE15 cells. Data are shown as means ± SEM (KD – knockdown, N = 3 – 10 experimental replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Staining, Immunofluorescence, Flow Cytometry, Fluorescence, Western Blot, Knockdown

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 7 The role of PICT1 in nuclear DNA damage and mitochondrial (mt) function. Increased PICT1/TRIM22 interaction induced by smoking leads to decreased PICT1 levels and high ROS production. This caused nuclear and mtDNA damage, common deletions, mitochondrial superoxide generation, and reduced mtDNA amount and respiration, contributing to ATII cell death and emphysema development

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III

doi: 10.1074/jbc.m808981200

Figure Lengend Snippet: FIGURE 1. Srx reduces sulfinic forms of mitochondrial Prx III in cells. HeLa and A549 cells were cultured under normal conditions (A). A549 cells were transfected with either pcDNA3 or pcDNA3-Srx (B and C) or with human Srx-specific siRNA or control RNA (D and E) and then cultured for 24 h. All cells were exposed to 200 M H2O2 for 10 min, washed with Hanks’ balanced salt solution, and cultured for the indicated times in culture media supplemented with 10% fetal bovine serum. Cell lysates were prepared and analyzed by SDS-PAGE (A, B, and D) or two-dimensional PAGE (C and E) followed by immunoblot analysis with antibodies specific to sulfinic 2-CysPrxs(Prx-SO2),Srx,PrxIII,and-tubulin.Theregiononthetwo-dimensionalimmunoblotscorrespondsto a molecular mass of 28 kDa and an isoelectric point of 5.9–6.4 characteristic of Prx III. The positions of oxidized (Ox) and reduced (Re) Prx III are indicated.

Article Snippet: Cell Culture and Establishment of Stable Cells—HeLa (human cervical carcinoma), HEK 293 (human embryonic kidney cells), and A549 (human lung carcinoma) cell lines were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Cell Culture, Transfection, Control, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III

doi: 10.1074/jbc.m808981200

Figure Lengend Snippet: FIGURE 2. Mitochondrial translocation of Srx under oxidative conditions. HeLa cells transfected with an expression vector for FLAG-tagged Srx (A and B) and normally grown A549 cells (C) were exposed to 200 M H2O2 for 10 min and then allowed to recover from oxidative stress as in Fig. 1. A and upper panel of C, after the indicated times, cell homogenates were separated into nuclear pellet and postnuclear supernatant (PNS). 0.5 ml of PNS was further separated into 0.5 ml of cytosolic (Cyto) and mitochondria-enriched heavy membrane (HM) fractions, and then the latter was prepared as 0.05 ml of lysates. Equal volumes (0.03 ml) of Cyto fractions and HM lysates were ana- lyzed by immunoblotting for the indicated proteins. Lower panel of C, the indicated percent volumes of aliquots from HM lysates (0.05 ml) or PNS (0.5 ml) were subjected to immunoblot analysis for Prx III. B, cells were stained for the FLAG epitope (green) and Prx III (red) and then examined by confocal microscopy.

Article Snippet: Cell Culture and Establishment of Stable Cells—HeLa (human cervical carcinoma), HEK 293 (human embryonic kidney cells), and A549 (human lung carcinoma) cell lines were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Translocation Assay, Transfection, Expressing, Plasmid Preparation, Membrane, Western Blot, Staining, FLAG-tag, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Increased Cx26 is positively correlated with gefitinib resistance in NSCLC cells. ( a ) Differential expression of Cx26, Cx31.1, Cx32, and Cx43 in different gefitinib-sensitive NSCLC cell lines was determined by RT-PCR. ( b and c ) High level of Cx26 in gefitinib-insensitive A549 and H1299 cells than that in gefitinib-sensitive HCC827 and PC9 cells was detected by RT-PCR and western blotting. GAPDH or β -actin was used as internal loading control

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 induces acquired gefitinib resistance in NSCLC cells via GJIC-independent manner. ( a ) Functional GJIC was detected by parachute assay and no detectable GJIC was found in HCC827 GR, PC9 GR, and their parental cells. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( b ) No enhancement of GJIC in these cells incubated with 10, 20, and 40 μ M of RA (a well-defined GJIC enhancer) for 4, 8, 12, 24, and 48 h, respectively. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( c and d ) Immunofluorescence staining of the cellular localization of Cx26 with or without RA treatment. All scare bars represent 50 μ m

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Functional Assay, Fluorescence, Incubation, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 and PI3K/Akt pathway functionally interplay to promote EMT and gefitinib resistance in NSCLC cells. ( a and b ) Effect of LY294002 or Akt overexpression on Cx26 expression in HCC827, PC9, and their GR cells was determined by western blotting. ( c ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on cell morphology changes in HCC827 and PC9 cells. Original magnification, × 400. ( d – f ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on the expression of EMT markers (E-cadherin, vimentin, and slug), cell migration, and invasion, as well as cell sensitivity to gefitinib in HCC827 and PC9 cells, respectively. Error bars are mean±S.D. from four independent experiments, ** P <0.01 versus vector group. # P <0.05 and ## P <0.01 versus Akt-overexpressing group

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Western Blot, Migration, Plasmid Preparation

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

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter.

doi: 10.1177/1010428319851014

Figure Lengend Snippet: Figure 5. Overexpression of RUNX2 in A549 cell line induced with TGF-b1 to promote EMT process. Human lung adenocarcinoma A549 cells were pre-cultured with or without 10 ng/mL TGF-b1 for 48 h. (a)–(c) E-CADHERIN, N-CADHERIN, and VIMENTIN mRNA levels in A549 cells. (d)–(f) RUNX2/p56 and RUNX2/p57 mRNA and protein levels in A549 cells with or without TGF-b1 treatment. Statistical analyses were performed with respect to A549 cells without TGF-b1. *p\0.05; **p\0.01; ***p\0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (ATCC ; Manassas, VA) was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 5% antibiotics (ampicillin and streptomycin) and incubated at 37 C in humidified 5% CO2 atmosphere.

Techniques: Over Expression, Cell Culture

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter.

doi: 10.1177/1010428319851014

Figure Lengend Snippet: Figure 6. Knockdown of RUNX2 affects EMT marker expression. A549 cells were stimulated with or without TGF-b1 as described in Figure 4. A549 cells were infected with lentiviral particles coding for shRNAs against RUNX2. Effective down-regulation was confirmed by qRT-PCR (a) and Western blot (b) analyses 72h post infection. TFIIB protein levels were used as loading control (c). E-CADHERIN, (d) N-CADHERIN, (e) VIMENTIN, (f) TWIST1, and (g) SNAIL1 mRNA levels were quantified by qRT-PCR 72h after infection. Statistical analyses were performed with respect to cells infected with virus generated with the pLKO.1 empty vector (sh-Ctrl.). *p\0.05; **p\0.01; ***p\0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (ATCC ; Manassas, VA) was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 5% antibiotics (ampicillin and streptomycin) and incubated at 37 C in humidified 5% CO2 atmosphere.

Techniques: Knockdown, Marker, Expressing, Infection, Quantitative RT-PCR, Western Blot, Control, Virus, Generated, Plasmid Preparation

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter.

doi: 10.1177/1010428319851014

Figure Lengend Snippet: Figure 7. Effect of RUNX2 knockdown on transwell migration and invasion assay. (a) A549 cells were stimulated with or without 10 ng/mL TGF-b1 and infected with lentiviral particles coding shRNAs against RUNX2. Cells penetrating the membrane were fixed and stained after 48 h as described in experimental procedures. Migrating cells were counted after additional incubation with 10 ng/mL TGF-b1 for 48 h. Data are shown as mean 6 SD, ***p\0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (ATCC ; Manassas, VA) was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 5% antibiotics (ampicillin and streptomycin) and incubated at 37 C in humidified 5% CO2 atmosphere.

Techniques: Knockdown, Migration, Invasion Assay, Infection, Membrane, Staining, Incubation

Journal: Cells

Article Title: Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

doi: 10.3390/cells12131757

Figure Lengend Snippet: Figure 2. Effects of conditioned medium (CM) obtained from A549 cells treated with MAGL inhibitor JZL184 (A), MAGL substrate 2-AG (B), additional MAGL inhibitors JW651 (C) and MJN110 (D), or DAGL inhibitor RHC 80267 (E) on migration, viability, and tube formation of HUVECs. Migration (Boyden chamber assay, white bars), viability (WST-1 assay, black bars), and tube formation (tube formation assays, gray bars) of HUVECs were determined after incubation with CM from A549 cells for 24 h (migration and viability assay) or 2 h (tube formation analysis). The CMs used were from A549 cells previously incubated for 48 h with vehicle (Veh) or the indicated concentrations of the test compounds. All percentage values given refer to serum-free DMEM (unconditioned medium, UCM) set to 100%. Data represent mean ± SEM of n = 6 ((A,B,E), migration and tube formation), n = 3–4 (C,D), or n = 8 ((E), viability) per group. ** p ≤0.01, *** p ≤0.001 vs. UCM; # p ≤0.05, ## p ≤0.01, ### p ≤0.001 vs. CM of vehicle-treated A549 cells; one-way ANOVA with Bonferroni post hoc test.

Article Snippet: A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; DSMZ cat. No. ACC-107, RRID:CVCL_0023).

Techniques: Migration, Boyden Chamber Assay, WST-1 Assay, Incubation, Viability Assay

Journal: Cells

Article Title: Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

doi: 10.3390/cells12131757

Figure Lengend Snippet: Figure 3. Evaluation of the role of the endocannabinoid system in the antiangiogenic effect of JZL184 (A) and 2-AG (B) on HUVECs by testing the effect of AM-251 (CB1 antagonist), AM-630 (CB2 antagonist), and capsazepine (Capsa, TRPV1 antagonist) on the antiangiogenic effect of CM from A549 cells treated with 1 µM JZL184 (A) or 2-AG (B). A549 cells were pre-incubated with the respective receptor antagonist (all tested at a final concentration of 1 µM) for 1 h and then incubated with vehicle, JZL184, or 2-AG for 48 h before CM was collected. Migration (Boyden chamber assay, white bars), viability (WST-1 assay, black bars), and tube formation (tube formation assays, gray bars) of HUVECs were determined after incubation with CM from A549 cells for 24 h (migration and viability assay) or 2 h (tube formation analysis). The control experiment shown in (C) demonstrates the effect of CM of A549 cells previously incubated with AM-251, AM-630, and capsazepine alone for 48 h on HUVEC migration, viability, and tube formation. In the experiment shown in (D), modulation of angiogenic properties by CM of A549 cells previously treated for 48 h with vehicle, JZL184 (1 µM), palmitic acid (PA, 10 µM), or the combination of JZL184 (1 µM) and PA (10 µM) was determined. PA was used to supplement potentially reduced free fatty acids due to MAGL inhibition. All percentage values given refer to HUVECs suspended in vehicle-containing CM set to 100%. Data represent mean ± SEM of n = 9 (A), n = 3 ((B,D), tube formation), n = 6 ((C), migration, tube formation; (D), migration), n = 7 ((C), viability), or n = 4 ((D), viability) per group. * p ≤0.05, *** p ≤0.001 vs. CM of vehicle-treated A549 cells; # p ≤0.05, ## p ≤0.01, ### p ≤0.001 vs. CM of JZL184- or 2-AG-treated A549 cells; one-way ANOVA with Bonferroni (A,B,D) or Dunnett (C) post hoc test.

Article Snippet: A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; DSMZ cat. No. ACC-107, RRID:CVCL_0023).

Techniques: Incubation, Concentration Assay, Migration, Boyden Chamber Assay, WST-1 Assay, Viability Assay, Control, Inhibition

Journal: Cells

Article Title: Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

doi: 10.3390/cells12131757

Figure Lengend Snippet: Figure 4. Effect of JZL184 (A,B) and 2-AG (C,D) on TIMP-1 protein expression in A549 cells and angiogenic abilities of HUVECs suspended in conditioned medium (CM) of vehicle- or JZL184- treated A549 cells in the presence or absence of TIMP-1 siRNA. In (A,C), the concentration-dependent effects of JZL184 (A) and 2-AG (C) on TIMP-1 protein expression are shown. A549 cells were incubated with vehicle and the respective concentration of JZL184 or 2-AG for 48 h followed by Western blot analysis. All percentage values given refer to vehicle-treated cells set to 100%. Data (A,C) represent mean ± SEM obtained from densitometric analysis of n = 4 experiments per group. In (B,D), A549 cells were incubated with transfection reagent in the absence of any siRNA (first and second triplets or bands) and transfected with TIMP-1 siRNA (TIMP-1 si; third and fourth triplets or bands) or non-silencing siRNA (nonsi; fifth and sixth triplet or bands) for 24 h in 10% FCS containing DMEM. Thereafter, A549 cells were washed and treated with vehicle, 1 µM JZL184 (C), or 1 µM 2-AG (D) for 48 h in serum-free DMEM prior to collection of CM. Migration (Boyden chamber assay, white bars), viability (WST-1 test, black bars), and tube formation (tube formation assays, gray bars) of HUVECs was measured after suspension in CM from A549-treated cells. The incubation time of HUVECs was 24 h for migration and viability testing and 2 h for tube formation analysis. Monitoring of TIMP-1 protein was performed in parallel using CM obtained from A549 cells. The Western blot images shown here are representative of a total of three (B) or four (D) experiments performed. For analysis of angiogenesis data in (B,D), vehicle-containing CM was set at 100%. Data represent mean ± SEM of n = 3 ((B), migration, tube formation; (D)) or n = 4 ((B), viability) per group. ** p ≤0.01, *** p ≤0.001 vs. corresponding vehicle control; ## p ≤0.01, ### p ≤0.001 vs. the respective JZL184 (B) or 2-AG (D) group without siRNA; one-way ANOVA with Dunnett (A,C) or Bonferroni (B,D) post hoc test.

Article Snippet: A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; DSMZ cat. No. ACC-107, RRID:CVCL_0023).

Techniques: Expressing, Concentration Assay, Incubation, Western Blot, Transfection, Migration, Boyden Chamber Assay, Suspension, Control

Journal: Cells

Article Title: Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

doi: 10.3390/cells12131757

Figure Lengend Snippet: Figure 5. Effect of conditioned medium (CM) derived from another lung cancer cell line (H358) and from a non-cancerous bronchial epithelial cell line (BEAS-2B) on the angiogenic capacities of HUVECs. Migration (Boyden chamber assay, white bars), viability (WST-1 assay, black bars), and tube formation (tube formation assays, gray bars) of HUVECs were determined after suspension in CM from vehicle-, JZL184-, and 2-AG-treated H358 (A,B) or BEAS-2B cells (C,D). To generate CM, H358 or BEAS-2B cells were incubated for 48 h with vehicle (Veh) or the indicated concentrations of test compounds. The exposure time of HUVECs to the indicated CM was 24 h for the migration and viability assay and 2 h for tube formation analysis. Vehicle-containing UCM was set at 100%. Data represent mean ± SEM of n = 3 ((A–D), tube formation; (A–C), migration; (A), viability), n = 4 ((D), migration), n = 5 ((B), viability), or n = 6 ((C), viability) per group. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 vs. UCM; # p ≤0.05, ## p ≤0.01, ### p ≤0.001 vs. CM of vehicle-treated A549 cells; one-way ANOVA with Bonferroni post hoc test.

Article Snippet: A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; DSMZ cat. No. ACC-107, RRID:CVCL_0023).

Techniques: Derivative Assay, Migration, Boyden Chamber Assay, WST-1 Assay, Suspension, Incubation, Viability Assay

Journal: Cells

Article Title: Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

doi: 10.3390/cells12131757

Figure Lengend Snippet: Figure 7. Impact of MAGL inhibitor JZL184 on the growth of A549 xenografts from nude mice as well as on the number of CD31- (angiogenesis and vascularization marker) and TIMP-1-positive cells in xenografts. Tumors were generated by subcutaneous inoculation of 1 × 107 A549 cells into the right dorsal flank. Animals were treated with either vehicle or JZL184 (4, 8, or 16 mg/kg i.p.) every 72 h for 28 days. Tumor size was measured with an external caliper and calculated as described in Materials and Methods and are shown as tumor volumes over time in panel A. The images beside the time-course were taken from representative tumors on day 28. Quantification of CD31 (B) and TIMP-1 (C) was performed by counting the positively stained cells of xenograft-derived paraffine sections stained with the indicated antibodies and calculating the percentage relative to the total number of cells per field of view. Values are means ± SEM of n = 7–8 (A), n = 4 (B), or n = 5 (C) animals per group. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 vs. vehicle; one-way ANOVA with Dunnett post hoc test.

Article Snippet: A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; DSMZ cat. No. ACC-107, RRID:CVCL_0023).

Techniques: Marker, Generated, Staining, Derivative Assay

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 1 N‐linked glycosylation of RBD protein is required for binding to ACE2. (a) Binding activity of SARS‐CoV‐2 RBD and S2 proteins derived from HEK293 cells or Escherichia coli. Binding was measured using an ACE2 binding assay, and nucleocapsid protein (N) and HIV p24 (P24) were used as negative controls. (b) Binding activity of RBD expressed either in HEK293 cells or in E. coli to titrated concentrations of ACE2. (c) Detection of purified and deglycosylated recombinant RBD and ACE2 proteins by SDS‐PAGE. From left to right: untreated proteins, proteins deglycosylated (dg) under native conditions, proteins deglycosylated under reducing conditions. (d) Detection of RBD and ACE2 binding using the ACE2 binding assay under conditions where either or both RBD and ACE2 were deglycosylated (dg). (e) RBD was deglycosylated with individual enzymes: PNGase F, O‐glycosidase, α2‐3,6,8,9 neuraminidase A, β1‐4 galactosidase S, or β‐N‐acetylhexosaminidasef, prior to the ACE2 binding assay. Statistical significance was tested by T test for all experiments. ****p < 0.0001. n ≥3 for all experiments.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Activity Assay, Derivative Assay, Recombinant, SDS Page

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 2 Glycosylation of RBD at Asn343 is essential for interaction with ACE2. (a) Detection of the binding of RBD wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives to ACE2 using the ACE2 binding assay. 40 ng/mL ACE2 was used in all assays. (b) Immunoblot analysis of recombinant SARS‐CoV‐2 RBD wild type or N331Q, N343Q, and N331Q/N343Q double mutated derivatives expressed within purified pseudovirus particles propagated in BHK21 cultured cells. (c) Detection of luciferase activity in A549 cultured cells infected with pseudovirus harboring S1 wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives. (d) Detection of luciferase activity in Vero cultured cells infected with pseudovirus harboring S1 wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives. (e) ACE2 expression levels (protein) in four cell lines, NIH3T3, A431, A549, and Vero, were detected by immunoblot. (f) The ACE2 binding was measured in the presence of blocking antibodies, including anti‐RBD (1H9; 4A9; 1F9), anti‐ S2, and anti‐N, to assess the specificity of ACE2 binding to RBD. Statistical significance was tested by T test for all experiments. ****p < 0.001. n = 4/5 for all experiments.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Western Blot, Recombinant, Cell Culture, Luciferase, Activity Assay, Infection, Expressing, Blocking Assay

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 3 Identification of glycan binding epitope in the N‐Glycan mutant site of RBD protein. (a) Identification of glycan recognition domains in the N331Q and N343Q mutants of RBD protein using a glycan array. Data represents two independent experiments. Data were analyzed using unpaired Tukey's test *p < 0.02 for statistical significance. (b) Schematic model of interaction inhibition in between double mutant (N331Q and N343Q) RBD mutant and WT RBD protein. Adapted from BioRender.com (2021). (c) N‐Glycan distribution on sites N90 and N322 of human ACE2 receptor, which are proximal to the RBD binding region of S1 protein (red arrows show the glycans with terminal galactose). Modified from Shajahan and colleagues, with permission.23

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Mutagenesis, Inhibition

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 6 Proposed model: Mutations in N‐glycosylation sites at N331 and N343 residues of RBD protein inhibit S‐ACE2 binding, IL‐6 expression and cytotoxicity. I confirm and agree with the possibility that my figure images might be selected and used as a journal cover image at issue publication.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Expressing