Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 1. Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, human samples of normal urothelium (N ¼ 10) and urothelial carcinoma of the bladder (N ¼ 8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma (UCC) compared with the normal urothelium. B, upregulation of these genes was validated by 2-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N ¼ 3) and normal urothelium (N ¼ 3). Ten of 13 genes (asterisked) showed statistical significance (t test; P < 0.05) for differential expression in urothelial carcinoma compared with normal urothelium.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 2. MLN8237 (MLN) induces cell-cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 showed specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, PI staining with flow cytometry analysis was conducted to assess cell-cycle changes. T24, UM-UC- 3, and RT4 cells were treated with 10 nmol/L to 1 mmol/L MLN8237 for 48 hours. All 3 cell lines showed dramatic cell-cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also showed a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Cytometry

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 3. Cellular phenotypes of T24 and RT4 cells differ after MLN8237 (MLN) treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells show a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic Aurora A expression, whereas RT4 cells lacked cytoplasmic Aurora A expression. E, real-time imaging of T24 and RT4 cells treated with MLN8237 was conducted over 48 hours. T24 cells exhibited dramatic increases in cell size as a result of repeated cell-cycle progressions without separation of daughter cells. RT4 cells seemed to become arrested after one failed mitotic attempt, preventing repeated cell-cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Microscopy, Expressing, Imaging

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 4. MLN8237 (MLN) induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 hours. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 and 45 nmol/L, respectively) and lowest potency in RT4cells (IC50 of 120 nmol/L). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 hours after initiation of treatment. C, annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 hours after initiation of MLN8237 treatment. D, clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nmol/L MLN8237.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Cytometry, Inhibition

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 6. Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 hours (left), or sequentially, with one drug for 48 hours, followed by washout and the other drug for 48 hours (middle and right). MTS assay was used to quantify the effect on cell viability of these combination treatments. MLN8237 showed synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right), and antagonistic effects when dosed simultaneously (left).

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: Scientific Reports

Article Title: Quinoline carboxylic acid derivatives as potent ectonucleotidase inhibitors

doi: 10.1038/s41598-026-36994-1

Figure Lengend Snippet: ( A ) Fluorescence spectroscopy and microscopy analysis of the antagonist drug molecule 4d. ( A ) Fluorescence spectroscopy profiles of 4d , excited between 410 and 500 nm, with corresponding emissions between 400 and 600 nm. ( B ) The bright-field image of A549 cells; Fluorescence microscopy image (red channel) of A549 cells treated with compound 4d; Merged image combining bright-field and fluorescence channels, highlighting the localization of the compound 4d on the cell membrane.

Article Snippet: Fluorescence imaging experiments were performed using A549 human lung carcinoma cells from American Type Cultures Collection (ATCC, Manassas, VA), while untreated A549 cells were used as controls in fluorescence imaging to account for background autofluorescence and confirm the specificity of compound 4d uptake and signal.

Techniques: Fluorescence, Spectroscopy, Microscopy, Membrane

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: ApoSense : a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

doi: 10.1007/s00259-005-1905-x

Figure Lengend Snippet: Concomitant confocal imaging of HeLa cell undergoing apoptosis by DDC (green fluorescence) and annexin V (red fluorescence). DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external membrane

Article Snippet: Human adult T-cell leukemia Jurkat cells (clone E6-1) and cervical carcinoma HeLa cells (CCL-2.2), were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37°C in 5% CO 2 .

Techniques: Imaging, Fluorescence, Membrane

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT is a novel centrosomal component. (A) Colocalization of UXT with the centrosome marker protein γ-tubulin. U2OS cells stably expressing EGFP:UXT (green) were labeled with anti-γ-tubulin (red). Images of cells in the G1, S/G2, prometaphase, metaphase, and anaphase are shown. (B) GFP-UXT is localized to the spindle poles during mitosis. U2OS cells stably expressing EGFP:UXT (green) were stained with an anti-α-tubulin (red) antibody. (C) The FLAG:UXT protein is localized to the centrosome. U2OS cells transfected with FLAG:UXT were immunostained with the mouse anti-FLAG antibody (red) and the rabbit anti-γ-tubulin antibody (green).

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Marker, Stable Transfection, Expressing, Labeling, Staining, Transfection

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Characterization of the anti-UXT antibodies. (A) The anti-UXT antibody 1B2 can detect both the endogenous UXT and the UXT fusion proteins. Cell lysates from HEK293T cell (lane 1), HEK293T cells transfected with EGFP:UXT (lane 2), and HEK293T cells transfected with FLAG-tagged UXT (lane 3) were separated by 15% SDS-PAGE. The expression of UXT was examined with the anti-UXT antibody 1B2 by Western blot analysis. Note that the anti-UXT antibody 1B2 can detect the endogenous UXT protein (18KD), EGFP:UXT (50KD), and FLAG:UXT (25KD). (B and C) Immunofluorescence detection of the endogenous UXT proteins in U2OS cells using the anti-UXT antibodies. U2OS cells were fixed with methanol and stained with anti-γ-tubulin antibody (green) in combination with either the anti-UXT antibody 15A6 (B, in red) or the anti-UXT antibody 6D3 (C, in red). DNA was stained with DAPI (blue). The arrows indicate the centrosomes.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Transfection, SDS Page, Expressing, Western Blot, Immunofluorescence, Staining

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Overexpression of UXT disrupts the centrosome structure. (A) Loss of centrosomal γ-tubulin staining in U2OS cells after overexpression of EGFP:UXT. U2OS cells with transient expression of EGFP:UXT (green) were immunostained with the anti-γ-tubulin antibody (red). DNA was stained with DAPI. Note that the γ-tubulin staining on the centrosome is diminished in cells with over expression of GFP-UXT. (B) Electron microscopic image of disorganized centrosome in U2OS cells with overexpression of EGFP: UXT. U2OS cells, transfected with either EGFP or GFP-UXT, were processed for electron microscope imaging. Top, image of the centrosome in U2OS cells transfected with EGFP (control). Bottom, image of the abnormal centrosome in U2OS cells transfected with EGFP:UXT. The arrow indicates a normal centriole.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Staining, Expressing, Transfection, Microscopy, Imaging, Control

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT siRNA knockdown causes cell death. Two nonoverlapping UXT siRNAs were tested and produced similar results. The data obtained with one UXT siRNA were shown. The data obtained with another UXT siRNA were shown as supplemental data (Figure S2). (A) Efficacy of siRNA knockdown of the endogenous UXT protein. U2OS cells were treated with transfection reagent (NT), nonspecific siRNA (NS siRNA), or siRNA for UXT (UXT siRNA), respectively. The cell lysates were subject to Western blot using either the anti-UXT antibody 1B2 (top) or the anti-β-tubulin antibody (bottom). The protein levels of UXT were specifically reduced after 72-h treatment with siRNA for UXT. (B) UXT knockdown inhibits cell proliferation. U2OS cells were treated with nonspecific siRNA (left) or siRNA for UXT (right). Pictures were taken 72 h later. (C) UXT knockdown leads to cell death. U2OS cells were treated with nonspecific RNA or the UXT siRNA oligos. Seventy-two hours later, all the cells were collected and used for propidium iodide staining of DNA content by fluorescence-activated cell sorting. (D) p53 is not required for cell death caused by UXT knockdown. The HCT116 (p53+/+) or p53-negative HCT116 (p53-/-) cells were treated with either the control siRNA or the UXT siRNA. Seventy-two hours after transfection, the percentage of cell death was assessed by the trypan blue assay. The diagram shows the representative results of three experiments.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Knockdown, Produced, Transfection, Western Blot, Staining, Fluorescence, FACS, Control

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Generation of p53null A549 cells by CRISPR/Cas9 technology. (A) Single-guide RNA (sgRNA) sequence designed to target p53WT for gene knockout by CRISPR/Cas9 technology. (B) Western blot screening of p53 protein expression in CRISPR A549 cell clones. (C) Western blot analysis confirmed loss of p53 protein expression in p53null cell clones A549-KO1, A549-KO2, and A549-KO3. Cells were pretreated with either Nutlin-3 or 5-fluorouracil to induce p53 accumulation in A549 cells. (D) Caspase 3/7 assay indicated that all p53null CRISPR clones had lower caspase activity compared with p53WT A549 cells in response to 4 h treatment of Nutlin-3 (25 µM, left panel) or 5-fluorouracil (50 µg/ml, right panel). * p < 0.05. (E) WST-1 cell proliferation assay indicated that the three selected p53null CRISPR clones were resistant to Nutlin-3 (15 µM, left panel) or 5-fluorouracil (30 µg/ml, right panel) induced growth inhibition/cell death.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: CRISPR, Sequencing, Gene Knockout, Western Blot, Expressing, Clone Assay, Activity Assay, Proliferation Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: p53null A549 cells exhibit decreased influenza virus susceptibility compared with parental p53WT A549 cells. (A) p53null cells A549-KO1, A549-KO2, and A549-KO3 and p53WT A549 were infected with human influenza virus (IAV) (strain A/Puerto Rico/8/1934 H1N1), 24 h later, cells were fixed, permeabilized, and labeled for influenza nucleoprotein (NP) detection by flow cytometry. The population of NP-positive cells was gated, and their percentages are shown. (B) Percentages of NP-positive cells 24 h post-IAV infection were compared between A549 and A549-KO3 cells from seven individual experiments. *** p < 0.001. (C) Flow cytometry analysis of NP-positive p53WT A549 and A549-KO3 cells 24 h post-IAV infection at a range of multiplicity of infection (MOI). (D) Fluorescence imaging of A549 and A549-KO3 cells co-labeled for viral NP (green) and cellular p53 (red) 24 h post-IAV infection. Scale bar, 50 µm. (E) Real-time quantitative PCR (RT-qPCR) measurement of viral RNA encoding NP, hemagglutinin (HA), and non-structural protein 1 (NS1) in mock- and IAV-infected A549 and A549-KO3 cells at various time points. (F) RT-qPCR measurement of viral RNA encoding NP, HA, and NS1 in culture supernatants of IAV-infected A549 and A549-KO3 cells at various time points.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Virus, Infection, Labeling, Flow Cytometry, Fluorescence, Imaging, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Decreased IAV susceptibility is not mediated by blocked cell entry at the initial stage of infection or decreased caspase 3 activation in p53null cells. (A) Real-time quantitative PCR (RT-qPCR) of viral RNA detected in IAV-infected A549, A549-KO2, and A549-KO3 cells at 1.5 h post-infection [multiplicity of infection (MOI) = 0.001]. (B) Caspase 3/7 activity in mock- or IAV-infected A549 and p53null cells. * p < 0.05. (C) Caspase 3/7 activity in mock- or IAV-infected A549 and A549-KO3 cells in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk (40 µM). * p < 0.05. (D) Flow cytometry analysis of IAV nucleoprotein (NP) expression in mock- or IAV-infected A549 and A549-KO3 cells, in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk. (E) RT-qPCR of viral RNA in the culture supernatant of IAV-infected A549 and A549-KO3 cells, in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk. (F) Cellular cytotoxicity in mock- and IAV-infected p53WT and p53null A549 cells. (G) Cell viability in mock- and IAV-infected p53WT and p53null A549 cells. For panels (B–G) , all measurements were performed at 24 h post-IAV infection (MOI = 0.001).

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Transcriptome analysis of mock- and IAV-infected A549 and A549-KO3 cells. (A) Venn diagram of significantly differentially expressed genes in A549-KO3 PR8 versus A549-KO3 Mock (blue circle) and A549 PR8 versus A549 Mock (red circle). (B) Heatmap showing the 396 overlapping genes significantly differentially expressed in both A549-KO3 PR8 versus A549-KO3 Mock, and A549 PR8 versus A549 Mock. (C) Venn diagram of significantly differentially expressed genes in A549-KO3 Mock versus A549 Mock (green circle) and A549-KO3 PR8 versus A549 PR8 (yellow circle). (D) Heatmap showing the 720 overlapping genes significantly differentially expressed in both A549-KO3 Mock versus A549 Mock, and A549-KO3 PR8 versus A549 PR8. (E) Venn diagram of significantly differentially expressed genes in A549-KO3 PR8 versus A549-KO3 Mock (blue), A549 PR8 versus A549 Mock (red), A549-KO3 Mock versus A549 Mock (green), and A549-KO3 PR8 versus A549 PR8 (yellow). (F) Heatmap showing the 50 overlapping genes significantly differentially expressed in all four groups.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Interferon-induced transmembrane proteins (IFITMs) expression is highly induced by IAV infection and interferons in p53null cells compared with p53WT A549 cells. (A) Expression levels of the top 3 most differentially expressed genes (IFITM1, IFITM2, and IFITM3) from the 50 overlapping gene set (Figure F) were validated by real-time quantitative PCR (RT-qPCR) validation in IAV-infected A549 and A549-KO3 cells [multiplicity of infection (MOI) = 0.001] at the indicated time points. (B) Western blot analysis of expression of viral nucleoprotein (NP), p53, IFITM1, and IFITM2/3 in p53WT A549 and p53null A549-KO3 cells 24 h post-IAV infection at a range of MOI. β-actin blotting was used as a loading control. (C) RT-qPCR analysis of IFITM1, IFITM2, and IFITM3 expression level in mock- or IAV-infected p53WT A549, p53null A549-KO1, A549-KO2, and A549-KO3 cells (MOI = 0.001) at 24 h post-infection. * p < 0.05. (D) Immunofluorescence microscopy images of IFITM1 and IFITM2/3 expression in IAV-infected p53WT A549, p53null A549-KO1, A549-KO2, and A549-KO3 cells (MOI = 0.001) at 24 h post-infection. Scale bar, 20 µm. (E) RT-qPCR analysis of IFITMs mRNA expression level in interferon-β 1a (IFN-β-1a) treated p53WT A549 and p53null A549-KO3 cells pretreated with Nutlin-3 or DMSO solvent control. * p < 0.05. (F) Western blot analysis of IFITM1, IFITM2/3, p53, and p21 protein expression level in IFN-β-1a treated p53WT A549 and p53null A549-KO3 cells pretreated with Nutlin-3 or DMSO solvent control. β-actin blotting was used as a loading control. For panels (E,F) , both A549 and A549-KO3 cells were treated with Nutlin-3 (25 µM) or DMSO for 6 h, followed by IFN-β-1a treatment (50 U/ml) for another 24 h.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Control, Immunofluorescence, Microscopy, Solvent

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Knockdown of interferon-induced transmembrane proteins (IFITMs) in p53null A549 cells restores susceptibility to IAV infection. A549-KO3 cells were transfected with either IFITM1 (si-IFITM1), IFITM2 (si-IFITM2), IFITM3 (si-IFITM3), a combination of three short interfering RNAs (siRNAs) (si-IFITM1/2/3), or scramble control siRNA (si-Ctrl). 24 h later, cells were detached using trypsin and re-plated onto cell culture plates or μ-slides and after overnight culture, cells were infected with IAV (multiplicity of infection = 0.001) or mock control. 24 h post-infection, cells were harvested for different assays and the RNA and protein lysates were also collected. A549 cells transfected with control siRNA were also included as a benchmark reference. (A) Real-time quantitative PCR analysis of IFITM1, IFITM2, IFITM3, IAV nucleoprotein (NP), IAV hemagglutinin (HA), and IAV non-structural protein 1 (NS1) gene expression level in IAV-infected cells pretreated with different siRNAs. For IFITM1, IFITM2, and IFITM3, expression data were normalized to that of the reference gene HPRT, and then to control siRNA transfected A549-KO3 cells, while for NP, HA, and NS1 gene expression, data were normalized to an HPRT control. * p < 0.05. (B) Western blot analysis of IAV NP, IFITM1, and IFITM2/3 protein expression in IAV-infected p53WT A549 and p53null A549-KO3 cells pretreated with different siRNAs. β-actin blotting was used as a loading control. (C) Flow cytometry analysis of Mock- or IAV-infected p53WT A549 and A549-KO3 cells pretreated with different siRNAs. Each population of NP-positive cells was boxed and their percentages were shown. (D) Representative immunofluorescence microscopy images of IAV-infected A549-KO3 cells pretreated with different siRNAs. IAV NP was detected using a FITC-labeled anti-NP antibody, and cell nuclei were stained with Hoechst 33342. Scale bar, 50 µm. (E) Percentages of NP-positive cells from IAV-infected A549-KO3 cells pretreated with different siRNAs. Five areas were randomly selected for image capture and the% of NP-positive cells was calculated by the number of NP-positive cells (green) as a proportion of total cell numbers, reflected by Hoechst staining (blue). * p < 0.05.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Knockdown, Infection, Transfection, Control, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Microscopy, Labeling, Staining

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Overexpressed interferon-induced transmembrane proteins (IFITMs) reduce cellular susceptibility to IAV infection. Mammalian expression plasmids encoding human IFITM1, IFITM2, or IFITM3 were constructed and transfected individually into p53WT A549 cells. After 24 h, cells were detached using trypsin and re-plated onto cell culture plates or μ-slides and after overnight culture, cells were infected with IAV (multiplicity of infection = 0.001) or mock control. At 24 h post-infection, cells were harvested for different assays and protein lysates were also collected. (A) Transfection efficiency was measured by flow cytometry analysis using IFIMT1- or IFITM2/3-specific antibodies. (B) Overexpression of IFITMs in transfected A549 cell cultures was detected by Western blot. (C) Representative immunofluorescence microscopy images of IAV-infected A549 cell cultures after transfection with IFITM1, IFITM2, or IFITM3 expression plasmids. IAV nucleoprotein (NP) is shown in green, and Hoechst 33342-stained nuclei in blue. Scale bar, 50 µm. (D) Percentage of NP-positive cells in IAV-infected A549 cell cultures that overexpressed IFITM1, IFITM2, or IFITM3 proteins, quantified from fluorescence microscopy images. Five areas were randomly selected for image capture and the percentages of NP-positive cells (green) were calculated relative to the total cell numbers, reflected by Hoechst staining (blue). ** p < 0.01. (E) Immunofluorescence microscopy images of IAV-infected A549 cells transfected with IFITM1, IFITM2, or IFITM3 expression plasmids. IFITMs are shown in red, IAV NP in green, and Hoechst 33342-stained nuclei in blue. Scale bar: 20 µm.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Expressing, Construct, Transfection, Cell Culture, Control, Flow Cytometry, Over Expression, Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: p53 regulates interferon-induced transmembrane proteins (IFITMs) independent of its transcriptional activity. (A) Schematic presentation of modular structures of full-length p53 and short isoform Δ40p53 proteins. (B) Western blot analysis of p53 (detected by antibody FL393, sc-6243), IFITM1, and IFITM2/3 expression levels in interferon-β 1a (IFN-β-1a) treated p53WT A549, A549-KO3, and A549-Δ40 cells. β-actin blotting was used as a loading control. (C) Real-time quantitative PCR (RT-qPCR) analysis of IFITMs mRNA expression in IFN-β-1a treated p53WT A549, A549-KO3, and A549-Δ40 cells. * p < 0.05. (D) A549, A549-KO3, and A549-Δ40 were infected with IAV [multiplicity of infection (MOI) = 0.001] and after 24 h were analyzed for IAV nucleoprotein (NP) expression by flow cytometry. The populations of NP-positive cells were boxed and their percentages shown. (E) Percentage of NP-positive cells in IAV- (MOI = 0.001) -infected p53WT A549, A549-KO3, and A549-Δ40 cells quantified from fluorescence microscopy images: between 4 and 6 areas were randomly selected for image capture and the percentages of NP-positive cells within the total cell population were calculated. * p < 0.05. (F) Western blot analysis of p53 (detected by both DO-1, sc-126 and FL393, sc-6243), IAV NP, IFITM1, and IFITM2/3 protein expression in p53WT A549, A549-KO3, and A549-Δ40 cells following IAV infection (MOI = 0.001). β-actin blotting was used as a loading control. (G) RT-qPCR measurement of viral RNAs encoding NP, hemagglutinin (HA), and non-structural protein 1 (NS1) genes detected either in IAV- (MOI = 0.001) infected cells (top) or from the culture supernatant (bottom). * p < 0.05. (H) RT-qPCR analysis of IFITM1, IFITM2, and IFITM3 gene expression in mock- or IAV-infected p53WT A549, A549-KO3, and A549-Δ40 cells. * p < 0.05.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Flow Cytometry, Fluorescence, Microscopy, Gene Expression