Journal: Cell Reports

Article Title: B.1.617.2 enters and fuses lung cells with increased efficiency and evades antibodies induced by infection and vaccination

doi: 10.1016/j.celrep.2021.109825

Figure Lengend Snippet: The spike protein of SARS-CoV-2 B.1.617.2 promotes efficient entry into human lung and colon cells, causes more cell-to-cell fusion, and evades antibody-mediated neutralization (A) Schematic overview of the S protein from SARS-CoV-2 variant B.1.617.2. RBD, receptor-binding domain; TD, transmembrane domain. (B) Location of the mutations found in SARS-CoV-2 variant B.1.617.2 in the context of the trimeric spike protein (color code: light blue, S1 subunit with RBD in dark blue; gray, S2 subunit; orange, S1/S2 and S2′ cleavage sites; red, mutated amino acid residues). (C) Pseudotyped particles bearing the S protein of wild-type (WT) SARS-CoV-2 or variant B.1.617.2 were inoculated onto the indicated cell lines, and transduction efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates at 16 to 18 h post transduction. Presented are the average (mean) data from 6 biological replicates (each conducted with technical quadruplicates) for which transduction was normalized against SARS-CoV-2 S WT (= 1). Error bars indicate the standard error of the mean (SEM). Statistical significance of differences between WT and B.1.617.2 S proteins was analyzed by two-tailed Student’s t test (p > 0.05, not significant [ns]; ∗∗ p ≤ 0.01). See also Figure S1 A. (D) BHK-21 expressing the S protein of WT SARS-CoV-2 or variant B.1.617.2 were subsequently incubated with soluble ACE2 (harboring a C-terminal Fc-tag derived from human IgG) and AlexaFluor-488-conjugated anti-human antibody before being subjected to flow cytometry. ACE2 binding efficiency was analyzed by measuring the geometric mean channel fluorescence at 488 nm. Untransfected cells and cells transfected with empty expression plasmid served as controls. Presented are the average (mean) data from 6 biological replicates (each conducted with single samples). Error bars indicate the standard deviation (SD). Statistical significance of differences between WT and variant B.1.617.2 S proteins was analyzed by two-tailed Student’s t test (p > 0.05, ns). (E) Analysis of S protein-induced cell-to-cell fusion. A549-ACE2 cells were transfected with expression plasmid for the indicated S proteins or empty vector (EV). At 24 h post transfection, cells were fixed and subsequently stained with May-Gruenwald and Giemsa solutions. Presented are representative microscopic images (scale bar, 200 μm). For quantification of fusion efficiency, the total number of nuclei in syncytia per image was counted. Presented are the average (mean) data from 4 biological replicates (each conducted with single samples; for each sample, 3 randomly selected areas were imaged and independently analyzed by 2 persons). Error bars indicate the SEM. Statistical significance of differences between WT and B.1.617.2 S proteins was analyzed by two-tailed Student’s t test ( ∗∗∗ p ≤ 0.001). (F) Neutralization of particles bearing SARS-CoV-2 WT or B.1.617.2 S proteins by monoclonal antibodies used for COVID-19 therapy. Pseudotyped particles bearing the S protein of WT SARS-CoV-2 or variant B.1.617.2 were incubated for 30 min at 37°C in the presence of escalating concentrations (0.00002, 0.0002, 0.002, 0.02, 0.2, and 2 μg/mL) of the indicated SARS-CoV-2 S protein-specific monoclonal antibody (please see Figure S1 B) or an unrelated control antibody (please see Figure S1 C) before being inoculated onto Vero cells. Transduction efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates at 16 to 18 h post transduction. Presented are the average (mean) data from a single biological replicate (conducted with technical quadruplicates) for which transduction was normalized against samples that did not contain any antibody (= 0% inhibition). Error bars indicate the SD. The results were confirmed in a separate experiment. (G) Neutralization of particles bearing SARS-CoV-2 WT, B.1.351, or B.1.617.2 S proteins by antibodies in convalescent plasma. Pseudotyped particles bearing the S protein of SARS-CoV-2 WT, B.1.351 or B.1.617.2 were incubated for 30 min at 37°C in the presence of different dilutions of convalescent plasma (1:25, 1:100, 1:400, 1:1,600, 1:6,400, and 1:25,600). Transduction efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates at 16 to 18 h post transduction and used to calculate the plasma dilution factor that leads to 50% reduction in S protein-driven cell entry (neutralizing titer 50 [NT50]). Presented are the data from a single biological replicate (conducted with technical quadruplicates) for a total of 8 convalescent plasma (black lines indicate the median; error bars indicate the SEM). Statistical significance of differences between the indicated groups was analyzed by Kruskal-Wallis analysis with Dunn’s post hoc test (p > 0.05, ns; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001). Please see also Figure S1 D. (H) The experiment was performed as described for (G), but this time serum from Comirnaty/BNT162b2-vaccinated individuals was investigated. Presented are the data from a single biological replicate (conducted with technical quadruplicates) for a total of 15 vaccinee sera (black lines indicate the median; error bars indicate the SEM). Please see also Figure S1 E.

Article Snippet: 293T (human, female, kidney; ACC-635, DSMZ; RRID: CVCL_0063), Vero76 cells (African green monkey kidney, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Maisner), BHK-21 (Syrian hamster, male, kidney; CCL-10, ATCC; RRID: CVCL_1915, kindly provided by Georg Herrler) and A549-ACE2 cells , which were derived from parental A549 cells (human, male, lung; CRM-CCL-185, ATCC; RRID:CVCL_0023; kindly provided by Georg Herrler), were cultured in Dulbecco’s modified Eagle medium (PAN-Biotech) supplemented with 10% fetal bovine serum (FBS, Biochrom), 100 U/ml penicillin and 0.1 mg/ml streptomycin (pen/strep) (PAN-Biotech).

Techniques: Neutralization, Variant Assay, Binding Assay, Transduction, Virus, Luciferase, Activity Assay, Two Tailed Test, Expressing, Incubation, Derivative Assay, Flow Cytometry, Fluorescence, Transfection, Plasmid Preparation, Standard Deviation, Staining, Bioprocessing, Control, Inhibition, Clinical Proteomics

Journal: Cell Reports

Article Title: B.1.617.2 enters and fuses lung cells with increased efficiency and evades antibodies induced by infection and vaccination

doi: 10.1016/j.celrep.2021.109825

Figure Lengend Snippet:

Article Snippet: 293T (human, female, kidney; ACC-635, DSMZ; RRID: CVCL_0063), Vero76 cells (African green monkey kidney, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Maisner), BHK-21 (Syrian hamster, male, kidney; CCL-10, ATCC; RRID: CVCL_1915, kindly provided by Georg Herrler) and A549-ACE2 cells , which were derived from parental A549 cells (human, male, lung; CRM-CCL-185, ATCC; RRID:CVCL_0023; kindly provided by Georg Herrler), were cultured in Dulbecco’s modified Eagle medium (PAN-Biotech) supplemented with 10% fetal bovine serum (FBS, Biochrom), 100 U/ml penicillin and 0.1 mg/ml streptomycin (pen/strep) (PAN-Biotech).

Techniques: Produced, Virus, Clinical Proteomics, Recombinant, Plasmid Preparation, Software, Imaging, Binding Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549 cells were transduced to stably overexpress ACE2 (A549-A), DPP4 (A549-D) in the absence or presence of TMPRSS2 (A549-AT and A549-DT). ACE2 and DPP4 expression was confirmed by western blot (A) , while TMPRSS2 expression was confirmed using flow cytometry (B) . (C-D) A549-derived or Calu-3 cells were inoculated with lentiviral particles pseudotyped with the spike proteins of MERS-CoV (C) , VSV, SARS-CoV-1, WIV1-CoV, WIV16-CoV, SARS-CoV-2 Hu1, SARS-CoV-2 delta or SARS-CoV-2 omicron (D) or pseudoparticles lacking envelope protein (no env) (C-D) for 2 h, then incubated for an additional 72 h, at which point luciferase activity was measured to assess pseudoparticle entry. The data are expressed as fold change relative to the luciferase signal obtained with no envelope. Graphs show mean +/- SEM from three independent experiments performed in triplicate.

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Derivative Assay, Incubation, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A) Schematic showing cell-surface TMPRSS2-mediated CoV entry compared to endosomal cathepsin-mediated entry. Camostat mesylate inhibits the TMPRSS2 pathway, while E64d inhibits the endosomal pathway. Schematic was prepared using Biorender.com . (B-C) A549-A or A549-AT cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with SARS-CoV-2pp (B) or SARS-CoV-1-like pseudoparticles (C) for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. Data are expressed as percentage relative to DMSO control. Graphs show mean +/- SEM from at least three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (**p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Infection, Incubation, Luciferase, Activity Assay, Control

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A) The presence of cell surface sialic acid was confirmed by flow cytometry using SNA, MAL-II and ECL lectins, which bind to α2,6-linked, α2,3-linked sialylated and galactosylated glycans, respectively. SNA and MAL-II bind is decreased upon loss of sialic acid; ECL binding is increased upon loss of sialic acid. (B-C) Soluble recombinant spike glycoprotein (SGP) binding to A549-A and A549-A CMAS KO cells assessed by flow cytometry using the CR3022 anti-spike antibody to measure SGP binding. Enzymatic desialylation was achieved by incubating cells with NanH for 30 min. Heparin inhibition was performed by pre-incubating SGP with heparin 10 min at 37°C prior to the addition to cells. Graphs show mean +/- SEM from an experiment performed in duplicate. Statistical significance was assessed by two-way ANOVA (****p<0.0001; ns, not significant).

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Flow Cytometry, Binding Assay, Recombinant, Inhibition

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A) A549-A WT or CMAS KO cells were inoculated with the indicated pseudoparticles for 2 h at 37°C, at which point the inocula were removed and cells were overlaid with complete media. After 72 h, lysates were collected to measure luciferase reporter activity to assess pseudoparticle entry. (B) A549-A WT or CMAS KO cells were pre-treated with DMSO vehicle or E64d (10 μM) for 1 h at 37°C, then inoculated with pseudoparticles as described above. Data are expressed as percentage relative to WT cells (A) or DMSO-treated control cells (B) . (A-B) Graphs show mean +/- SEM from at least three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (****p<0.0001; ns, not significant).

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Luciferase, Activity Assay, Control

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-D) A549-derived cells or Calu-3 cells were pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C. Cells were then washed and processed for fluorescence microscopy (A) or inoculated with SARS-CoV-2 pseudoparticles (B-D) . (A) NanH-treated cells were stained with SNA-FITC (binds sialic acid) or ECL-FITC (binds galactose) diluted to final concentration of 20 μg/mL in PBS, then washed with PBS and imaged by fluorescence microscopy (20X magnification; scale bar, 50 μm). Lectin staining confirmed removal of sialic acid by the NanH treatment. (B-D) NanH-treated cells were inoculated with the indicated SARS-CoV-2 pseudoparticles for 2 h at 37°C, then washed and incubated in complete media for 72 h. Luciferase activity was then measured to assess viral entry. Data are expressed as percentage relative to DMSO control. (B-D) Graphs show mean +/- SEM from at least three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (**p<0.01; ****p<0.0001; ns, not significant).

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Derivative Assay, Fluorescence, Microscopy, Staining, Concentration Assay, Incubation, Luciferase, Activity Assay, Control

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549-D, A549-DT or Calu-3 cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations (A) , or pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C (B) . Cells were then washed and inoculated with MERS-CoVpp for 2 h at 37°C prior to being washed and overlaid with complete media for 72 h. Luciferase activity was then measured to assess viral entry. Data are expressed as percentage relative to DMSO control. (C) A549-A or A549-AT cells were pre-treated with NanH as above (B) , then washed and infected with HCoV-OC43 (MOI 0.5) for 1 h at 37°C. Cells were then incubated in complete media for an additional 7 h, at which point they were fixed and processed for immunofluorescence microscopy using an antibody against the HCoV-OC43 N protein. Representative images are shown (20X magnification; scale bar, 50 μm). (C) The percentage of infected cells in each condition was determined using ImageJ. (A-C) Graphs show mean +/- SEM (A-B) or SD (C) from three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: Parental A549 cells (BEI Resources #NR-52268) were cultured in Ham’s F-12 K (Kaighn’s) medium (Fisher Scientific #21127030) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Techniques: Luciferase, Activity Assay, Control, Infection, Incubation, Immunofluorescence, Microscopy

Journal: Journal of Virology

Article Title: A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

doi: 10.1128/JVI.01715-20

Figure Lengend Snippet: Screening of SARS-CoV-2 reporter constructs. (A) A549-ACE2 cells were transduced with lentiviruses encoding the reporter constructs specified on the top left of each panel. Cells were fixed at 32 h postransduction and GFP localization was analyzed using a wide-field fluorescence microscope. (B) Quantification of images acquired as in panel A. The percentages of nuclear or cytosolic GFP are shown (green and gray, respectively). At least 290 cells were counted for each construct. (C) Cells transduced as in panel A were infected with SARS-CoV-2 (MOI = 5) at 16 h postransduction. Cells were fixed at 16 hpi and dsRNA (magenta) and the GFP-based reporter construct (green) were detected by immunofluorescence using a wide-field fluorescence microscope. (D) Quantification of images acquired as in panel C. Percentages of infected cells positive for dsRNA only (magenta) or double positive for nuclear GFP signal and dsRNA (orange) are shown. For each construct, more than 85 cells were counted. Scale bars = 50 μm. (E) A549-ACE2 cells stably expressing reporter construct 14 were infected with SARS-CoV-2 (MOI = 10). Cells were fixed at the indicated time postinfection (top left corner) and the SARS-CoV-2 N protein was stained by IF. N protein (magenta) and GFP (green) subcellular distributions were determined by confocal microscopy. Scale bar = 50 μm. (F) Quantification of the acquired images as described for panel E. Percentages of cells with N protein staining (magenta) and nuclear GFP signal (green) are shown for the different time points. For each time point more than 190 cells were analyzed.

Article Snippet: Both the A549-ACE2 C2 reporter clone and the parental A549-ACE2 cells without reporter construct expression were incubated with serial dilutions of remdesivir for 30 min and infected with SARS-CoV-2 (MOI = 5).

Techniques: Construct, Transduction, Fluorescence, Microscopy, Infection, Immunofluorescence, Stable Transfection, Expressing, Staining, Confocal Microscopy

Journal: Journal of Virology

Article Title: A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

doi: 10.1128/JVI.01715-20

Figure Lengend Snippet: Application of the SARS-CoV-2 reporter cell line for live cell imaging of viral infection and assessment of antiviral activity of remdesivir. (A) Experimental setup to monitor GFP-reporter activation in SARS-CoV-2-infected cells. (B) A549-ACE2-RC cells (clone C2) stably expressing the reporter construct 14 were infected with SARS-CoV-2 (MOI = 10). At two hours postinfection (hpi), live cell imaging was performed for 18 h using a confocal spinning disc microscope. Images of representative fields of view and time points are displayed. Scale bar = 50 μm. (C) A549-ACE2 and A549-ACE2-RC cells (clone C2) expressing the SARS-CoV-2 reporter construct 14 were incubated with remdesivir (1.1 μM) or DMSO only and infected with SARS-CoV-2 (MOI = 5). After 16 h, cells were fixed and stained for N protein prior to imaging with a confocal spinning disc microscope. Scale bar = 50 μm. (D) IC 50 calculation of remdesivir in reporter cell clone 2 infected with SARS-CoV-2 (MOI = 5). Percentages of inhibition were calculated by quantification of the number of N-positive cells and cells with nuclear GFP signal in duplicate wells for each compound concentration. Values were normalized by setting the average number of infected cells in the DMSO-treated sample as 0% inhibition.

Article Snippet: Both the A549-ACE2 C2 reporter clone and the parental A549-ACE2 cells without reporter construct expression were incubated with serial dilutions of remdesivir for 30 min and infected with SARS-CoV-2 (MOI = 5).

Techniques: Live Cell Imaging, Infection, Activity Assay, Activation Assay, Stable Transfection, Expressing, Construct, Microscopy, Incubation, Staining, Imaging, Inhibition, Concentration Assay

Journal: F1000Research

Article Title: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.150684.2

Figure Lengend Snippet: Summary of the cell lines used.

Article Snippet: Parental and TGM2 KO A549 cells were obtained from Abcam ( ).

Techniques:

Journal: F1000Research

Article Title: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.150684.2

Figure Lengend Snippet: Lysates of A549 (WT and TGM2 KO) were prepared and 40 μg of protein were processed for western blot with the indicated TGM2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab109121** at 1/1000, ab109200** at 1/10000, ab2386* at 1/500, ab310333** at 1/1000, ab421 at 1/500, ABCD_AI748** at 1/10, A21184** at 1/10000, ARP47471 at 1/500, ARP47472 at 1/500, AF4376 at 1/400, MAB4376* at 1/200, 3557** at 1/500, GTX111702 at 1/500, 15100-1-AP at 1/6000, 68006-1-Ig* at 1/10000, MA5-32819** at 1/500, MA5-12739* at 1/200. Predicted band size: 77 kDa. *Monoclonal antibody, **Recombinant antibody.

Article Snippet: Parental and TGM2 KO A549 cells were obtained from Abcam ( ).

Techniques: Western Blot, Staining, Membrane, Recombinant

Journal: F1000Research

Article Title: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.150684.2

Figure Lengend Snippet: A549 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated TGM2 antibodies pre-coupled to Dynabeads protein A or protein. Samples were washed and processed for western blot with the indicated TGM2 antibody. For western blot, A21184** was used at 1/10000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; n/s=non-specific signal. *Monoclonal antibody, **Recombinant antibody.

Article Snippet: Parental and TGM2 KO A549 cells were obtained from Abcam ( ).

Techniques: Immunoprecipitation, Western Blot, Staining, Recombinant

Journal: F1000Research

Article Title: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.150684.2

Figure Lengend Snippet: A549 WT and TGM2 KO cells were labelled with a green or a deep-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated TGM2 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and deep-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, which was the case for all antibodies tested, except ab310333**, ABCD_AI748 and A21184**, we tested antibodies at using the dilutions listed below. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab109121** at 1/1000, ab109200** at 1/300, ab2386* at 1/1000, ab310333** at 1/500, ab421 at 1/1000, ABCD_AI748** at 1/1000, A21184** at 1/1000, ARP47471 at 1/500, ARP47472 at 1/250, AF4376 at 1/100, MAB4376* at 1/100, 3557** at 1/500, GTX111702 at 1/1000, 15100-1-AP at 1/300, 68006-1-Ig* at 1/500, MA5-32819** at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

Article Snippet: Parental and TGM2 KO A549 cells were obtained from Abcam ( ).

Techniques: Staining, Concentration Assay, Microscopy, Recombinant

Journal: F1000Research

Article Title: A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.150684.2

Figure Lengend Snippet: Summary of the TGM2 antibodies tested.

Article Snippet: Parental and TGM2 KO A549 cells were obtained from Abcam ( ).

Techniques: Concentration Assay, Recombinant, Purification

Journal: Translational Cancer Research

Article Title: FOXO3/TRIM22 axis abated the antitumor effect of gemcitabine in non-small cell lung cancer via autophagy induction

doi: 10.21037/tcr.2019.12.33

Figure Lengend Snippet: TRIM22 was significantly upregulated in gemcitabine-resistant non-small lung cancer cells and corresponding transplanted tumor tissues of nude mice. (A) Expression Profiles of deregulated mRNAs between gemcitabine (Gem)-resistant non-small lung cancer Calu3 cells and parental cells by analyzing the GEO data set GSE 6914 (P-adjust <0.05, log2 change-fold >1 or log2 change-fold <−1). Different genes were selected for cluster analysis and R package heatmap was selected. Genes with similar expression patterns may have common functions or participate in common metabolic and signaling pathways. In the thermogram, red indicates high expression, while blue indicates low expression. (B) TRIM22 was significantly upregulated in GEM-resistant Calu3 cells (R) comparing with parental Calu3 cells (S) by analyzing the GSE6914. (C) Relative expression of TRIM22 in clinically collected patient serum samples and transplanted tumor tissues of nude mice detected using QPCR. Sensitive, serum samples from Gem-sensitive patient or transplanted tumors formed by inoculation of A549 cells; Resistant, serum samples from Gem-resistant patient or transplanted tumors formed by inoculation of A549/GR cells. *, P<0.05; **, P<0.01 vs. sensitive group; (D) relative expression of TRIM22 in A549/GR cells and A549 parental cells using QPCR, **, P<0.01 vs. A549 parental cells; (E) protein expression of TRIM22 in A549/GR cells and A549 cells detected using western blot.

Article Snippet: The NSCLC GR cell line A549/GR and its parental cell line A549 were purchased from Procell (Wuhan, China) and were cultured under the condition of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin sulfates) at 37 °C in humidified atmosphere of 5% CO 2 .

Techniques: Expressing, Protein-Protein interactions, Western Blot

Journal: Translational Cancer Research

Article Title: FOXO3/TRIM22 axis abated the antitumor effect of gemcitabine in non-small cell lung cancer via autophagy induction

doi: 10.21037/tcr.2019.12.33

Figure Lengend Snippet: TRIM22 overexpression could attenuate the sensitivity of A549 cells to GEM and its depletion could promote the sensitivity of A549/GR cells to GEM. (A) TRIM22 was successfully enhanced in Lv-TRIM22 lentivirus infected A549 cells and decreased in TRIM22-siRNA transfected A549/GR cells detected using QPCR (right) and western blot (left). (B) MTT assay was used to evaluate the cell viability after gemcitabine treatment at 24, 48 or 72 hours. *, P<0.05 vs. negative control cells; **, P<0.01 vs. negative control cells (negative control: Lv-Con in A549 cells and siRNA-NC in A549/GR cells); (C) cell cycle distribution of TRIM22 overexpressed A549 cells (LV-TRIM22) and TRIM22 silenced A549/GR cells (TRIM22-siRNA) following exposure to GEM. (D) Flow cytometry with annexin V FITC/PI double staining in detection of the GEM induced apoptosis of A549 cells, **, P<0.01 vs. negative control cells (negative control: Lv-Con in A549 cells and siRNA-NC in A549/GR cells). (E) Western blot assay was used to evaluate the apoptosis related proteins in A549 and A549/GR model cells after gemcitabine treatment at 24 hours.

Article Snippet: The NSCLC GR cell line A549/GR and its parental cell line A549 were purchased from Procell (Wuhan, China) and were cultured under the condition of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin sulfates) at 37 °C in humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Infection, Transfection, Western Blot, MTT Assay, Negative Control, Flow Cytometry, Double Staining

Journal: Translational Cancer Research

Article Title: FOXO3/TRIM22 axis abated the antitumor effect of gemcitabine in non-small cell lung cancer via autophagy induction

doi: 10.21037/tcr.2019.12.33

Figure Lengend Snippet: TRIM22 promoted GEM-induced pro-survival autophagy to protected NSCLC cells from apoptotic Death. TRIM22 could mediate the gemcitabine-induced LC3 puncta accumulation in A549 and A549/GR cells, the magnification was 400× (A) and the number of puncta in cells of different groups was counted and comparative analysis were made (B); **, P<0.01 vs. negative control cells (negative control: Lv-Con in A549 cells and siR-NA-NC in A549/GR cells); (C) Western blot assay was used to evaluate the autophagy re-lated proteins LC3 and P62 in A549 and A549/GR model cells after gemcitabine treatment at 24 h. (D) 3-MA (5 mM) and rapamycin were added to A549 and A549/GR cell cultures respectively for 1 h. prior to gemcitabine exposure, then the relative survival rate was esti-mated using MTT. *, P<0.05 vs. negative control cells; **, P<0.01 vs. negative control cells; negative control: Lv-Con in A549 cells and siRNA-NC in A549/GR cells; (E) 3-MA (5 mM) and rapamycin were added to A549 and A549/GR cell cultures respectively for 1 h. prior to gemcitabine treatment. Then cell apoptosis was detected using flow cytometry with annexin V FITC/PI double staining in model cells and the apoptotic rate of different treated cells was also analyzed statistically (F). *, P<0.05 vs. negative control cells; **, P<0.01 vs. negative control cells, (negative control: Lv-Con in A549 cells and siRNA-NC in A549/GR cells); (G) cell cycle distribution of TRIM22 overexpressed A549 cells (LV-TRIM22) and TRIM22 silenced A549/GR cells (TRIM22-siRNA) following expo-sure to GEM.

Article Snippet: The NSCLC GR cell line A549/GR and its parental cell line A549 were purchased from Procell (Wuhan, China) and were cultured under the condition of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin sulfates) at 37 °C in humidified atmosphere of 5% CO 2 .

Techniques: Negative Control, Western Blot, Flow Cytometry, Double Staining

Journal: Translational Cancer Research

Article Title: FOXO3/TRIM22 axis abated the antitumor effect of gemcitabine in non-small cell lung cancer via autophagy induction

doi: 10.21037/tcr.2019.12.33

Figure Lengend Snippet: TRIM22 expression was negatively transcriptional regulated by FOXO3. (A) There is potential transcriptional relationship between FOXO3 and TRIM22 promoter with the online programs JASPAR and ALGGEN-PROMO. (B) FOXO3 was significantly down-regulated in GEM-resistant Calu3 cells (R) comparing with parental Calu3 cells (S) by analyzing the GSE6914 (P-adjust <0.05, log2 change-fold >1 or log2 change-fold <−1). Different genes were selected for cluster analysis and R package heatmap was selected. Genes with similar expression patterns may have common functions or participation in common metabolic and signaling pathways. In the thermogram, red indicates high expression, while blue indicates low expression. (C) The protein expression of FOXO3 in A549/GR cells and A549 parental cells detected using western blot; (D) FOXO3 silence could decrease the p-FOXO3 but enhance the expression of TRIM22 and FOXO3 overexpression could increase the p-FOXO3 but decrease the TRIM22 expression in A549 and A549/GR cells respectively. (E) Bi-luciferase reporter assay as described in the section “Methods”. The luciferase level was significantly attenuated after co-transfection of FOXO3 with TRIM22-promoter. **, P<0.01 vs. TRIM22 promoter group. MTT assay was used to evaluate the A549/GR viability after gemcitabine treatment at 24, 48 or 72 hours (F), then cell survival rates in Lv-FOXO3 and LV-FOXO3-TRIM22 groups were compared and statistically analyzed, *, P<0.05 vs. FOXO3 overexpressed A549/GR cells (Lv-FOXO3); The cell apoptosis was detected using flow cytometry with annexin V FITC/PI double staining (G), and apoptotic rate of Lv-FOXO3 and LV-FOXO3-TRIM22 groups was compared and statistically analyzed, **, P<0.01 vs. Lv-NC cells, # , P<0.05 vs. FOXO3 overexpressed A549/GR cells (Lv-FOXO3).

Article Snippet: The NSCLC GR cell line A549/GR and its parental cell line A549 were purchased from Procell (Wuhan, China) and were cultured under the condition of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin sulfates) at 37 °C in humidified atmosphere of 5% CO 2 .

Techniques: Expressing, Protein-Protein interactions, Western Blot, Over Expression, Luciferase, Reporter Assay, Cotransfection, MTT Assay, Flow Cytometry, Double Staining

Journal: Respiratory Research

Article Title: Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

doi: 10.1186/s12931-021-01840-7

Figure Lengend Snippet: Circ_PIP5K1A was upregulated in DDP-resistant NSCLC tissues and cells. A IC 50 of DDP was examined by MTT assay in A549, A549/DDP ( A ) and H460, H460/DDP ( B ) cells. C , D the circ_PIP5K1A expression was assayed by RT-qPCR in tumor-resistant tissues ( C ) and DDP-resistant cells ( D ). E – H total RNA was treated with RNase R ( E , F ) and cells were incubated with Actinomycin D ( G , H ), then circ_PIP5K1A and PIP5K1A quantification was conducted by RT-qPCR. ** P < 0.01, *** P < 0.001

Article Snippet: The parental NSCLC cell lines (A549, H460) and DDP-resistant cell lines (A549/DDP, H460/DDP) were purchased from BioVector NTCC Inc. (Beijing, China).

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

Journal: Respiratory Research

Article Title: Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

doi: 10.1186/s12931-021-01840-7

Figure Lengend Snippet: Circ_PIP5K1A knockdown inhibited DDP resistance and malignant behaviors in DDP-resistant NSCLC cells. Transfection of si-circ_PIP5K1A or si-NC was performed in A549/DDP and H460/DDP cells. A , B RT-qPCR was used to detect the levels of circ_PIP5K1A and PIP5K1A. C , D MTT assay was used to determine the IC 50 value of DDP. E – G colony formation assay ( E ) and MTT ( F , G ) were used to assess cell proliferation capacity and cell viability. H – J flow cytometry was applied to examine cell cycle progression ( H , I ) and apoptosis ( J ). K , L transwell assay was applied to measure cell migration ( K ) and invasion ( L ). *** P < 0.001

Article Snippet: The parental NSCLC cell lines (A549, H460) and DDP-resistant cell lines (A549/DDP, H460/DDP) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, MTT Assay, Colony Assay, Flow Cytometry, Transwell Assay, Migration

Journal: Respiratory Research

Article Title: Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

doi: 10.1186/s12931-021-01840-7

Figure Lengend Snippet: Downregulation of miR-493-5p counteracted the effects of si-circ_PIP5K1A on DDP-resistant NSCLC cells. Transfection of si-NC, si-circ_PIP5K1A, si-circ_PIP5K1A + anti-miR-NC or si-circ_PIP5K1A + anti-miR-493-5p was conducted in A549/DDP and H460/DDP cells. A the expression analysis of miR-493-5p was completed through RT-qPCR assay. B the determination of IC 50 was performed through MTT assay. C – E the evaluation of cell proliferation and viability was carried out through colony formation assay ( C ) and MTT ( D , E ). F – H the examination of cell cycle ( F , G ) and apoptosis ( H ) was implemented through flow cytometry. I , J the assessment of cell migration ( I ) and invasion ( J ) was performed through transwell assay. ** P < 0.01, *** P < 0.001

Article Snippet: The parental NSCLC cell lines (A549, H460) and DDP-resistant cell lines (A549/DDP, H460/DDP) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, MTT Assay, Colony Assay, Flow Cytometry, Migration, Transwell Assay

Journal: Respiratory Research

Article Title: Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

doi: 10.1186/s12931-021-01840-7

Figure Lengend Snippet: MiR-493-5p acted as a sensitizer of DDP and tumor inhibitor in DDP-resistant NSCLC cells by inducing ROCK1 downregulation. Transfection of miR-NC, miR-493-5p, miR-493-5p + pcDNA or miR-493-5p + ROCK1 was carried out in A549/DDP and H460/DDP cells. A , B ROCK1 expression was quantified using RT-qPCR and Western blot. C IC 50 of DDP was measured via MTT assay. D – F cell proliferation and viability were detected via colony formation assay ( D ) and MTT ( E – F ). G – I cell cycle ( G , H ) and apoptosis ( I ) were analyzed by flow cytometry. J , K cell migration ( J ) and invasion ( K ) were evaluated using transwell assay. *** P < 0.001

Article Snippet: The parental NSCLC cell lines (A549, H460) and DDP-resistant cell lines (A549/DDP, H460/DDP) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Colony Assay, Flow Cytometry, Migration, Transwell Assay

Journal: Respiratory Research

Article Title: Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

doi: 10.1186/s12931-021-01840-7

Figure Lengend Snippet: Circ_PIP5K1A regulated DDP sensitivity to NSCLC in vivo by the expression regulation of miR-493-5p and ROCK1. A , B tumor volume ( A ) and weight ( B ) were determined in DDP + lenti-NC, DDP + lenti-circ-PIP5K1A and DDP + sh-circ_PIP5K1A groups. C , D the levels of circ_PIP5K1A and miR-493-5p in tumor tissues were examined by RT-qPCR. E , F ROCK1 mRNA and protein detection was performed via RT-qPCR and Western blot in tumor tissues. G Ki67 and Cleaved caspase3 levels in tumor tissues were analyzed using IHC assay. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The parental NSCLC cell lines (A549, H460) and DDP-resistant cell lines (A549/DDP, H460/DDP) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Adipose Triglyceride Lipase Loss Promotes a Metabolic Switch in A549 Non–Small Cell Lung Cancer Cell Spheroids

doi: 10.1016/j.mcpro.2021.100095

Figure Lengend Snippet: 3D cultured ATGL-KO A549 cells grown on CAM form bigger tumors and display proteomic changes . A , CAM assay workflow. B , principal component analysis of ATGL-KO and control samples. C , volcano plot of the LFQ data after filtering for at least three valid values in at least one group; significance threshold Student t test p -value 0.05, FDR 5%, S0 0.5; proteins marked in red are addressed in more detail in the text. ATGL, adipose triglyceride lipase; ATGL-KO, ATGL knockout; CAM, chorioallantoic membrane; FDR, false discovery rate; LD, lipid droplet; LFQ, label-free quantitation.

Article Snippet: Briefly, A549 parental cells were transfected with either a plasmid for directed knockout of human ATGL (sc-401711; Santa Cruz Biotechnology) or a control plasmid (sc-418922; Santa Cruz Biotechnology) using Lipofectamine3000, generating ATGL-KO or corresponding control cells.

Techniques: Cell Culture, Chick Chorioallantoic Membrane Assay, Control, Knock-Out, Membrane, Quantitation Assay