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

Journal: Nature Communications

Article Title: Programmed cell removal by calreticulin in tissue homeostasis and cancer

doi: 10.1038/s41467-018-05211-7

Figure Lengend Snippet: Cell surface asialoglycans regulates CRT-mediated PrCR. a , b Treatment with neuraminidase led to the removal of sialic acids from the cell surface of HL60 cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Cell surface sialic acids were examined by staining with EBL (a) and MAL (b) by flow cytometry analysis. EBL, Elderberry Bark Lectin; MAL, Maackia Amurensis Lectin II. c , d Examination of cell surface CRT and PHA-L binding sites on cancer cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Recombinant CRT ( c ) and PHA-L ( d ) binding after treatment were measured by flow cytometry. e , f Phagocytosis of cancer cells with neuraminidase treatment. In vitro Phagocytosis assays were performed with HL60, K562, DLD-1, and SW620 cells treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu) as target cells. Mouse bone marrow-derived ( e ) and human peripheral blood monocyte-derived ( f ) macrophages were used for the assay. Phagocytosis was normalized to the maximal response in the experiments. n = 3. * P < 0.05, ** P < 0.01 ( t -test) for phagocytosis between heat inactivated neuraminidase (Δneu)- and neuraminidase (neu)-treated groups. Error bars represent standard deviation. g , h Effects of suppressing the expression of endogenous neuraminidases in cancer cells. Neu1–Neu4 gene knockout were performed with CRISPR in HL60 cells. In vitro Phagocytosis assays were performed with HL60 cells as target cells and mouse bone marrow-derived macrophages ( h ). Macrophages were treated with PBS (ctrl) or lipopolysaccharide (LPS). Neu4 knockout led to the decrease of cell surface CRT-binding sites and inhibited cancer cell phagocytosis. Phagocytosis was normalized to the maximal response in the experiments. n = 3. * P < 0.05, ** P < 0.01 ( t -test) for phagocytosis between ctrl and Neu4 KO groups. Error bars represent standard deviation

Article Snippet: Human cancer derived cell lines SW620, DLD1, HL60, K562, and murine macrophage/monocyte cell line J774 were obtained from ATCC.

Techniques: Staining, Flow Cytometry, Binding Assay, Recombinant, In Vitro, Derivative Assay, Standard Deviation, Expressing, Gene Knockout, CRISPR, Knock-Out

Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Schematic illustrating difference between classic synthetic lethality and our common genetic architecture . Synthetic lethality consists of many individual Y i functions. These functions are cell-type-specific models with single features. Our proposed common genetic architecture is hypothesized to connect these “private” functions with shared CERES features. A common genetic architecture has many redundant edges, and more interconnected nodes. More nodes suggest that more cell-type-specific phenotypes are predictable, and more edges suggest redundancy. b A network built from the aggregation of all multivariate models. Genes are represented as nodes and feature-target gene relations as edges. Colors represent distinct subnetwork communities that were identified by the Louvain method. c Network communities with (right) and without (left) nodes/edges involving functional CRISPR features for a single Louvain community, and a comparison with our hypothesis from ( a ). Edges are colored based upon the data source; and nodes are colored based on the model score (of top ten feature model) of the corresponding gene as target. d To quantitate the visual similarity between our hypothesis in ( a ) and the data in ( c ) across all Louvain communities, we examined the differences in the clustering coefficient, the average number of neighbors, and the network heterogeneity. e gprofiler2 plots examine the enrichment of functional categories. f Residual plot identifies GO terms that are more (residuals of −log10 P values >10) or less (residuals of -log10 P values < −10) enriched in predictor genes than in target genes. Dots represent shared GO terms among the 100 most significant terms in target and predictor gprofiler2 analysis result. The p- values from gprofiler2 for ( e ) and ( f ) are based on hypergeometric tests with multiple testing corrections using the g:SCS method. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Functional Assay, CRISPR, Comparison

Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Two separate pooled screens were performed in a cell line (PC9) that was not included in model training and validation. Experiment 1 was the full Brunello library. A 21-day dropout experiment was performed in PC9 cells. Measurements on 18,114 genes were direct and form the gold standard. The L200 can be computationally extracted from the full screen and compared to these gold-standard measurements. A new L200 standalone library of 800 guides targeting 200 genes was cloned. This library can be used to perform a small-scale lossy compression experiment. The data can then be compared to the gold standard. b Correlations of inferred vs measured CERES scores for both screens in ( a ) and a comparison of the predictions between the standalone sets and the computationally extracted L200 set in the Brunello library. c A Venn diagram describes the overlap in “Hits” in the 500 most differentially required genes for growth in PC9 cells. Both of the lossy compression screens from ( a ) and the gold-standard (measured) data are compared. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Biomarker Discovery, Clone Assay, Comparison