Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Schematic depiction of the 30 SARS-CoV-2 encoded proteins in the order they appear in the genome. The polyprotein ORF1a(b) is (auto)proteolytically cleaved into 16 non-structural proteins (Nsp, turquoise). The structural proteins (yellow) are Spike (S), Membrane (M), envelope (E) and nucleoprotein (N). The set is complemented by the accessory proteins (red) ORF 3a, 3b, 3c, 6, 7a, 7b, 8, 9b, 9c and 10. b-e , Schematic depiction of the assay setup (top panel) and heatmap (red = inhibition, blue = induction) depicting modulation of innate immune pathways by overexpression of indicated SARS-CoV-2 proteins. Stimuli of the immune pathways are indicated. (a, b) Readout by Luciferase reporter gene assay (color represents the mean of n=3) using indicated promotor constructs in HEK293T cells, (c) autophagosome measurement by quantification of membrane-associated GFP-LC3B in HEK293T-GFP-LC3B cells (color represents the mean of n=4) or (d) Quantification of ASC specks in THP-1 cell lines by flow cytometry; cell lines doxycycline-inducible expressing the indicated transgenes were treated with Bacillus anthracis PA and LFn-MxiH to stimulate NLRC4 inflammasomes (color represents the mean of n=2). The vector/control is set to 1 (white). SeV, Sendai Virus. Rapa, Rapamycin. BafA, Bafilomycin A1, PA, protective antigen of B. anthracis , MxiH, Needle protein of S. flexneri.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Inhibition, Over Expression, Luciferase, Reporter Gene Assay, Construct, Flow Cytometry, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Schematic depiction of the type-I IFN signaling pathway. b , Exemplary immunoblot analysis showing activation of type-I IFN signaling markers using whole cell lysates (WCLs) of HEK293T cells expressing indicated proteins and stimulated with IFN-β (1000 U/mL, 45 min). Blots were stained with anti-pSTAT1, anti-STAT1, anti-pSTAT2, anti-STAT2, anti-IFNAR, anti-strep II and anti-actin. c , Quantification of the band intensities in (b) for IFNAR normalized to the band intensities of actin. Bars represent mean of n=3±SEM. d , Quantification of the band intensities in (b) for phospho-STAT1 (pSTAT1) normalized to the band intensities of actin. Bars represent mean of n=3±SEM. e , Schematic depiction of autophagy. f , Exemplary immunoblot analysis showing autophagy activity markers using WCLs of HEK293T cells expressing indicated proteins. Blots were stained with anti-SQSTM1/p62, anti-LC3B-II, anti-Beclin-1, anti-ULK1, anti-strep II and anti-actin. g , Quantification of the band intensities in (f) for LC3B-II normalized to the band intensities of actin. Bars represent mean of n=3±SEM. h , Quantification of the band intensities in (f) for p62 normalized to the band intensities of actin. Bars represent mean of n=3±SEM. i , Exemplary confocal laser scanning microscopy images of autophagy activation via GFP-LC3B (green) puncta formation. Indicated strep II-tagged SARS-CoV-2 proteins (red) were overexpressed in HeLa GFP-LC3B cells (green). CQ, Chloroquine (4 h 10 μM) was used as a positive control. Nuclei, DAPI (blue). Scale bar, 25 μM. j , Quantification by area of GFP-LC3B puncta divided by cell number from the images in (i). Bars represent the mean of n=38-100 cells±SEM.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Western Blot, Activation Assay, Expressing, Staining, Activity Assay, Confocal Laser Scanning Microscopy, Positive Control

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Heatmap (red = downregulation, blue = upregulation) depicting the fold changes of cellular and viral proteins during overexpression of indicated single SARS-CoV-2 proteins in HEK293T cells or b , SARS-CoV-2 infection (MOI 1) of Caco-2 cells 24 or 48 h post infection as assessed by mass spectrometry. c , Scatter plots of log2 fold enrichment and p-value of the GO-Term ‘late endosome’ in protein sets regulated more than 4-fold upon expression of indicated viral protein (a) or SARS-CoV-2 infection (b). d , Quantification of co-localization by Pearson Correlation of Rab9 and indicated viral proteins in HeLa cells transiently transfected with the indicated viral protein and GFP-Rab9. Bars represent the mean of n=7-15 cells±SEM. e , Exemplary confocal microscopy images of HeLa cells transiently expressing indicated viral proteins (red) and a marker of late endosomes GFP-Rab9 (green). Cells were stained with anti-strep II (red). Nuclei, DAPI (blue). Scale bar, 10 μm. f , Exemplary confocal microscopy images of the quantification in (g) stained with anti-TGN46 (green) and anti-strep II (red). Nuclei, DAPI (blue). Scale bar, 10 μm. g , Pearson’s correlation indicating colocalization between TGN46 and the indicated viral proteins from the image in (f). Bars represent the mean of n=6 cells±SEM. h, Quantification of non-Golgi associated vesicles per cell as puncta/cell of (f). Bars represent the mean of n=15-25 cells ±SEM.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Over Expression, Infection, Mass Spectrometry, Expressing, Transfection, Confocal Microscopy, Marker, Staining

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a-c , Immune activation of type-I IFN induction (a), type-I IFN signaling (b) or autophagy (c) in the presence of indicated proteins (Nsp1, Nsp3, Nsp7, Ndsp15, M, N, ORF3a, ORF6, ORF7a) of SARS-CoV-2 (blue), RaTG13-CoV (purple) or SARS-CoV-1 (red) assessed by IFN-β-promotor luciferase reporter gene assays stimulated with Sendai Virus (SeV, a). ISRE-promotor luciferase reporter gene assays stimulated with IFN-β (1000 U/ml, b). Membrane-associated GFP-LC3B (c) (n=4±SEM). Vector induction set to 100% (black). Controls, RABV P, MeV V or TRIM32 (grey). Bars represent the mean of n=3±SEM (a,b) or n=4±SEM (c). d , Dose dependent effect of SARS-CoV-2, RaTG13-CoV or SARS-CoV-1 Nsp15 expression on IFN-β induction stimulated with SeV (24 h). Quantification by IFN-β promotor dependent luciferase reporter activity. Lines represent one individual replicate. e , Dose dependent effect of Nsp15 expression on IFN-β signaling in HEK293T cells, stimulated with IFN-β (1000 U/ml, 8 h). Quantification by ISRE promotor dependent luciferase reporter activity. Lines represent one individual replicate.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , SARS-CoV-2 N RNA in the supernatant of SARS-CoV-2 (MOI 0.05, 48h p.i.) infected Calu-3 cells that were left untreated and/or were treated with the indicated amounts of indicated IFNs or pro-inflammatory cytokines as assessed by qPCR. Lines represent the mean of n=2±SD. b , Correlation between average inhibition of the indicated innate immune signaling pathway and impact on replication of SARS-CoV-2 after treatment with the respective cytokine. r, Pearson’s correlation. c , SARS-CoV-2 N RNA in the supernatant of SARS-CoV-2 (MOI 0.05, 48h p.i.) infected Calu-3 cells that were left untreated and/or were treated with the indicated combinations of indicated IFNs (5 U/ml) or Rapamycin (125 nM) either 24 h before the infection (Pre-treatment) or 6 h post infection (Post-treatment). Dots represent individual experiments, line the mean. Fold reduction compared to control is indicated. d , Immunoblot analysis of the SARS-CoV-2 infection using the WCLs of Calu-3 cells in (c). Blots were stained with anti-SARS-CoV-2 S, anti-SARS-CoV-2 N, and anti-GAPDH.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Infection, Inhibition, Western Blot, Staining

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet:

Article Snippet: Library quantity, purity and size selection were assessed using Qubit fluorometer (Qubit DNA Assay kit, Invitrogen Q32854) and an Agilent Bioanalyzer (D5000 ScreenTape, Agilent 5067–5592) with acceptable library size distribution between 600 and 1150bp.

Techniques: Recombinant, Antibody Labeling, Staining, Cell Isolation, Control, Mass Cytometry, Sequencing, Software

Journal: Cell

Article Title: Orphan quality control shapes network dynamics and gene expression.

doi: 10.1016/j.cell.2023.06.015

Figure Lengend Snippet: Figure 1. UBR5 binds transcriptional regulators with connections to c-Myc (A) Identification of endogenous FLAGUBR5 partners from 293T and HeLa cells by affinity purification and mass spectrometry. (B) Validation of UBR5 interactors in HeLa cells by affinity purification and western blot analysis. (C) Depletion of KIF2A only reduces this protein in FLAGUBR5 affinity purifications from HeLa cells. (D) Depletion of SPT5 destabilizes and reduces UBR5 binding of SPT4 in HeLa cells. (E) Endogenous FLAGUBR5 was immunoprecipitated from HeLa cells expressing HASPT5. Eluates were purified on aHA-resin and probed for UBR5, SPT4, and MCRS1 by western blot analysis. (F) Endogenous UBR5 was precipitated from hESC lysate using specific antibodies and probed for co-purifying proteins by western blot analysis. (G) Most UBR5 partners have close links to the c-Myc/WDR5 complex. See also Figure S1.

Article Snippet: For immunoprecipitation of endogenous UBR5 from H1 hESCs, bait protein was purified using protein G-agarose resin (SigmaAldrich, 11719416001) coupled with mouse anti-UBR5 antibody (Santa Cruz Biotechnology, sc-515494), in lysis buffer for 1 h, rotating at 4 C.

Techniques: Mass Spectrometry, Biomarker Discovery, Western Blot, Binding Assay, Immunoprecipitation, Expressing

Journal: Cell

Article Title: Orphan quality control shapes network dynamics and gene expression.

doi: 10.1016/j.cell.2023.06.015

Figure Lengend Snippet: Figure 2. UBR5 degrades transcriptional regulators with connections to c-Myc (A) A flow-cytometry-based screen identifies candidate UBR5 substrates in HeLa cells. (B) Validation of the c-MycDTAD and MCRS1 targets in DUBR5 HeLa cells or cells with the deletion of the UBR5 HECT or UBA domain (blue: genetic manipulation; orange: chemical manipulation). (C) UBR5 targets are stabilized upon E1 enzyme inhibition with PYR41. (D) UBR5 targets are stabilized upon p97 inhibition with NMS873. (E) UBR5 targets are stabilized upon proteasome inhibition. (F) Endogenous UBR5 targets increase in DUBR5 HeLa cells, as determined by western blot analysis. (G) Stability of UBR5 substrates was determined in cells depleted of related E3 ligases. See also Figures S1 and S2.

Article Snippet: For immunoprecipitation of endogenous UBR5 from H1 hESCs, bait protein was purified using protein G-agarose resin (SigmaAldrich, 11719416001) coupled with mouse anti-UBR5 antibody (Santa Cruz Biotechnology, sc-515494), in lysis buffer for 1 h, rotating at 4 C.

Techniques: Cytometry, Biomarker Discovery, Enzyme Inhibition Assay, Inhibition, Western Blot

Journal: Cell

Article Title: Orphan quality control shapes network dynamics and gene expression.

doi: 10.1016/j.cell.2023.06.015

Figure Lengend Snippet: Figure 3. UBR5 ubiquitylates transcriptional regulators (A) Endogenous UBR5 was purified from HeLa cells and incubated with E1, UBE2D3, ubiquitin, and 35S-labeled targets produced by in vitro transcription/ translation (IVT/T). Substrate modification was analyzed by autoradiography (left panel: a silver-stained gel of UBR5). (B) 35S-labeled MCRS1 was incubated with UBR5, E1, UBE2L3, and ubiquitin. (C) UBR5, UBR5C2768S, and UBR5DUBA were incubated with 35S-labeled MCRS1. (D) 35S-labeled MCRS1 was incubated with UBR5, E1, UBE2D3, and ubiquitin mutants and analyzed for ubiquitylation as above. (E) 35S-labeled MCRS1-Ub4 was incubated with UBR5, E1, UBE2D3, and ubiquitin mutants. See also Figure S3.

Article Snippet: For immunoprecipitation of endogenous UBR5 from H1 hESCs, bait protein was purified using protein G-agarose resin (SigmaAldrich, 11719416001) coupled with mouse anti-UBR5 antibody (Santa Cruz Biotechnology, sc-515494), in lysis buffer for 1 h, rotating at 4 C.

Techniques: Incubation, Ubiquitin Proteomics, Labeling, Produced, In Vitro, Autoradiography, Staining

Journal: Cell

Article Title: Orphan quality control shapes network dynamics and gene expression.

doi: 10.1016/j.cell.2023.06.015

Figure Lengend Snippet: Figure 6. UBR5 provides orphan quality control (A) UBR5 was incubated with TAMRA-labeled MCRS1 degron, E1, UBE2L3, and ubiquitin. Ubiquitylation was monitored by fluorescence. (B) UBR5 ubiquitylated TAMRA-labeled SPT5 degron peptides. (C) UBR5, UBR5C2768S, and UBR5DUBA were incubated with the SPT5 degron, E1, UBE2L3, and ubiquitin. (D) BACH2 protects MAFF from UBR5-dependent degradation. The MAFF reporter was expressed in cells depleted of UBR5 and/or co-expressing BACH2, as indicated. MAFF stability was determined by flow cytometry. (E) The MCRS1 reporter is stabilized by CCDC85B. (F) The RUVBL2 reporter is stabilized by RUVBL1. (G) The OCT4 reporter is stabilized by SOX2. (H) BACH2 protects endogenous MAFF from UBR5-dependent degradation. 293T cells were depleted of BACH2 and/or UBR5, and endogenous MAFF was visualized by western blot analysis. (I) SOX2 protects endogenous OCT4 from UBR5-dependent degradation in H1 hESCs.

Article Snippet: For immunoprecipitation of endogenous UBR5 from H1 hESCs, bait protein was purified using protein G-agarose resin (SigmaAldrich, 11719416001) coupled with mouse anti-UBR5 antibody (Santa Cruz Biotechnology, sc-515494), in lysis buffer for 1 h, rotating at 4 C.

Techniques: Control, Incubation, Labeling, Ubiquitin Proteomics, Expressing, Cytometry, Western Blot

Journal: Cell

Article Title: Orphan quality control shapes network dynamics and gene expression.

doi: 10.1016/j.cell.2023.06.015

Figure Lengend Snippet: Figure 7. Transcription factor degradation establishes network dynamics (A) FLAGMAX was affinity purified using FLAG antibodies from wild-type or DUBR5 HeLa cells, and co-purifying c-Myc was detected by western blot analysis. (B) Endogenous MAX was precipitated from wild-type or DUBR5 HeLa cells, and co-purifying c-Myc was detected by western blot analysis. (C) Wild-type or DUBR5 293T cells expressing FLAGMAX and HAc-MycT58A were treated with cycloheximide. FLAGMAX was immunoprecipitated, and co-purifying proteins were detected by western blot analysis. (D) FLAGMAX was immunoprecipitated from wild-type or DUBR5 cells treated with oligomycin and antimycin A, and co-purifying proteins were detected by western blot analysis. (E) FLAGMCRS1 was precipitated from wild-type or DUBR5 293T cells exposed to cycloheximide, and co-purifying proteins were detected by western blot analysis. (F) FLAGMCRS1 was immunoprecipitated from wild-type or DUBR5 cells, and co-purifying proteins were detected by western blot analysis. (G) Orphan quality control establishes network dynamics required for gene expression. Transcription factors (TFs) in the c-Myc network are continuously pro- duced, form complexes with stabilizing partners (SPs), and drive gene expression. Following dissociation, UBR5 degrades one subunit to render complex disassembly irreversible. The stabilizing partner can be re-used, whereas the unstable subunit must be re-synthesized. This network architecture allows tran- scription factors to efficiently switch from early to late partners while remaining susceptible to stress-induced inhibition of gene expression. See also Figure S8.

Article Snippet: For immunoprecipitation of endogenous UBR5 from H1 hESCs, bait protein was purified using protein G-agarose resin (SigmaAldrich, 11719416001) coupled with mouse anti-UBR5 antibody (Santa Cruz Biotechnology, sc-515494), in lysis buffer for 1 h, rotating at 4 C.

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Gene Expression, Synthesized, Inhibition

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: Cells were treated with doxorubicin in 5% FBS medium for 72 hr. A. Cell response to doxorubicin. MCF-12A (wt p53), SW48 (wt p53), COLO 320DM (mutant p53 R248W; COLO), WiDr (mutant p53 R273H), SW48/TP53 (mutant p53 R273H), SW48-Dox and TP53-Dox (mutant p53 R273H) cells were treated with doxorubicin for 72 hr. *, p <0.001, compared to wt p53 cells (MCF-12A, SW48). B. Doxorubicin induced EMT in cancer cells carrying a p53 R273H mutant allele. Cells (passages 8-10) of SW48, SW48/TP53, SW48-Dox and TP53-Dox were grown in multi-chamber slides with 10% FBS medium for 48 hr. Red, Alexa Fluor 555–vimentin (VIM); green, Alexa Fluor 488–E-cadherin (ECAD); blue, DAPI–nucleus. Scale bar represents 50 μm in photomicrographs (200x magnification). C. Western blotting for EMT marker assessment. Equal amounts of extracted detergent-soluble proteins (50 μg protein/lane) were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with antibodies of E-cadherin (ECAD), vimentin (VIM) or GAPDH. Protein levels were represented by ratios (mean ± SD) of ECAD or VIM densities normalized against GAPDH from three blots. *, p <0.01 compared to SW48 cells; **, p <0.001 compared to SW48-Dox cells.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Mutagenesis, Western Blot, Marker, SDS Page

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: A. Wound healing of cancer cells. Scale bar represents 50 μm in photomicrographs (×100 magnification). *, p <0.001 compared to TP53 cells; **, p <0.001 compared to SW48-Dox or TP53-Dox cells treated with vehicle. B. GCS activities analyzed by HPLC. Cer, NBD C6-ceramide. GlcCer, NBD C6-glucosylceramide. *, p <0.001 compared to Vehicle of TP53-Dox cells. C. Ceramides analyzed by ESI/MS/MS. Cer, ceramide. *, ≥twofold higher than vehicle.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Tandem Mass Spectroscopy

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: Cells of SW48-Dox and TP53-Dox lines were separately treated with 5 μM PDMP in 5% FBS medium. A. Tumor sphere formation. Scale bar represents 50 μm in photomicrographs (100x magnification). *, p >0.001 compared to SW48-Dox with vehicle; **, p<0.001 compared to TP53-Dox with vehicle. B. CSCs. Cells were incubated with Alexa-Fluor488 CD44v6 and APC-CD133 antibodies and analyzed by using flow cytometry. The detected CD44v6 + /CD133 + cells (CSCs) are identified in the plots by enclosure with an ellipse (upper right), and compared with vehicle controls, as percentages of total cells in the adjacent bar graph.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Incubation, Flow Cytometry

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: Cells were treated with PDMP (5 μM, 6 days) followed by doxorubicin exposure (50 nM, 48 hr). Equal amounts of detergent-soluble proteins (50 μg/lane) were resolved by 4-20% SDS-PAGE and immunoblotted with corresponding primary and secondary antibodies, sequentially. A. Effects of doxorubicin-induction and PDMP on p53 and p53-responsive protein expression. B. p53 modulates iPSC factor expression in cancer cells exposed to Dox. PDMP inhibits ceramide glycosylation, thereby increasing cellular ceramide levels, which may favor wt p53 expression over that of the R273H missense mutant. C. Protein expression levels. Protein levels are presented here as ratios of their densities normalized against GAPDH from three Western blots. *, p<0.05 compared to parental (SW48, TP53) or Dox-induced sublines (SW48-Dox, TP53-Dox); **, p<0.05 compared to SW-48, or SW48-Dox and SW48-Dox PDMP. pp53, phosphorylated p53 (Ser15); β-Cat, β-catenin; TGF-β, transforming growth factor β. D. Immunostaining of ceramide and pp53. The scale bar represents 5 μm in photomicrographs (200x magnification).

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: SDS Page, Expressing, Glycoproteomics, Mutagenesis, Western Blot, Immunostaining

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: Cells of SW48 and SW48/TP53 (TP53) lines were subcutaneously inoculated into athymic nude mice. Low doses of doxorubicin (200 μg/kg and 300 μg/kg, per 6 days, i.p.) and PDMP (4.0 mg/kg, per 3 days, i.p.) were administered for 32 days (5 cases/group). A. SW48 tumor growth. B. TP53 tumor growth. *, p <0.01, compared to doxorubicin alone treatments. C. Tumor weight after treatments. *, p <0.01, compared to doxorubicin alone treatments. D. GCS activities in tumors after treatments. *, p<0.001, compared to SW48 tumors treated with Dox (200 μg/kg); **, p <0.001, compared to TP53 tumors treated with doxorubicin (200 μg/kg); #, p<0.001, compared to SW48 tumors treated with Dox and PDMP.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: A. Western blotting. Equal amounts of detergent-soluble proteins (50 μg/lane) extracted were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies. Protein levels in the bar graph are presented as mean±SD of their densities normalized against GAPDH from three blots. *, p <0.01 compared to SW48 tumors treated with Dox; **, p <0.01 compared to TP53 tumors treated with Dox; #, p<0.01 compared to SW48 tumors treated with Dox and PDMP. B. Immunostaining of pp53 and p21. The scale bar indicates 25 μm in photomicrographs (200x magnification). Red, Alexa Fluor 555-pp53; green, Alexa Fluor 488-p21; blue, DAPI-nucleus. The scale bar indicates 50 μm in photomicrographs (200x magnification).

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, SDS Page, Immunostaining

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: A. Flow cytometry analysis of colon CSCs (CD133+/CD44V6) from tumors in mice treated with doxorubicin (Dox, 200 μg/kg, per 6-days, i.p.) alone or combined with PDMP (4.0 mg/kg, per 3-days, i.p.), for 32 days. *, p<0.001 compared to SW48 tumors treated with Dox; **, p<0.001 compared to TP53 tumors treated with Dox. B. Western blotting of pluripotency regulators in tumors. Equal amounts of detergent-soluble proteins (50 μg/lane) extracted were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies. Protein levels are represented as mean ± SD of their densities normalized against GAPDH from three blots. *, p <0.01 compared to Dox treatments (200 μg/kg); **, p <0.01 compared to SW48 tumors.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Flow Cytometry, Western Blot, SDS Page

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: After 48 h pretreatments with PDMP (5 μM), PDMP plus FB1 (25 μM), MBO-asGCS (100 nM), siRNA-p53 (100 nM), or siRNA-SC (100 nM), TP53-Dox cells were cultured in medium containing doxorubicin (50 nM) with each of these agents for 48 h to induce DNA damage and 72 h for cell viability assays. For the Western blot studies, the cells pretreated under each of the various sets of conditions, the doxorubicin (50 nM) exposure was combined with continuation of the various pretreatment agents for an additional 48 h to induce DNA damage before protein extraction. A. Western blotting. Equal amounts of detergent-soluble proteins extracted (50 μg/lane) were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies; representative blots are presented. GCS, glucosylceramide synthase; pp53, phosphorylated p53 (Ser15); β-cat, β-catenin; siRNA-SC, siRNA scrambled control. B. Ceramide affects wt p53 expression. Protein levels are represented as mean ± SD of their densities normalized against GAPDH from three settings of blots. *, p<0.001 compared to vehicle or siRNA-SC; **, p<0.001 compared to PDMP or MBO-asGCS treatment. C. Cell responses to doxorubicin. D. IC 50 values for doxorubicin. *, p<0.001 compared to siRNA-SC or vehicle. **, p<0.001 compared to PDMP or MBO-asGCS treatment.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Cell Culture, Western Blot, Protein Extraction, SDS Page, Control, Expressing

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: When mutant-heterozygous (HZ) cancer cells are exposed to a sub-lethal dose of doxorubicin (Dox), DNA damage induces overexpression of p53 R273H, and the presence of this missense mutant protein upregulates Zeb1 and β-catenin (β-Cat) stem-like factors. The consequent gain-of-function (GOF) presents as epithelial-mesenchymal transition (EMT) and induced pluripotency of stem cells (iPSCs), lending to tumor growth. Inhibition of glucosylceramide synthase (GCS)-catalyzed ceramide glycosylation with PDMP increases cellular ceramide levels, bring about restoration of wt p53 protein expression, in turn increasing p53-responsive proteins, including p21 and Puma, thereby suppressing tumor progression.

Article Snippet: Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Mutagenesis, Over Expression, Inhibition, Glycoproteomics, Expressing

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Ascorbic acid improves the cognitive level of aged mice and increases the number of CD8 + cells in aged mice. A Trajectory tracking map of mouse movement in the open field test, illustrating time spent in the central zone (seconds), average speed during movement episodes (cm/s), and total distance traveled within the central area. B Statistical chart of New Object Cognition Index. C Flow cytometry was utilized to analyze immune cell populations, including CD3 + T, CD4 + T, CD8 + T, CD11b + , and B cells, in the whole blood of middle-aged mice assigned to either the control group or the AA group ( D ). E Statistical analysis of the flow cytometry data was presented in a histogram format, with significance levels denoted as follows: ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: AA facilitates the early differentiation of T cells while concurrently suppressing myeloid differentiation. A A schematic depicting the early and long-term differentiation of T cells in an OP9-DL1 and lin-CD117 + HSC co-culture system is presented. B The diagram illustrates the progression of T cell maturation from CD44 and CD25 negative to positive selection. C Flow cytometry analysis and statistical bar graphs are utilized to demonstrate the AA-promoted changes in T cell DN stages, including the DN1 phase (CD44 + CD25 − ), DN2 phase (CD44 + CD25 + ), DN3 phase (CD44 − CD25 + ), and DN4 phase (CD44 − CD25 − ). D Flow cytometry analysis is utilized to examine the inhibition of myeloid differentiation, as well as to assess the long-term T cell differentiation promoted by AA in vitro ( E ). F and G Statistical bar graphs are generated to represent the flow cytometry results of CD8 + and CD4 + T cells, with significance levels denoted as ns: not significant ( P > 0.05), * ( P < 0.05), and ** ( P < 0.01), ( n = 3 for each group)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Co-Culture Assay, Selection, Flow Cytometry, Inhibition, Cell Differentiation, In Vitro, Generated

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: The Myh9 protein exhibits binding affinity towards AA and affects the proliferation of CD8 + T cells. A MetPro protein-metabolite interaction experimental workflow diagram. B Predicted binding differential protein KEGG pathway enrichment diagram. C Mass spectrometry predicted binding differential protein heatmap. D Protein interaction average degree analysis diagram. E Protein interaction network analysis diagram. F Concentration gradient binding curves of AA with Rhoa and Myh9 highlight the binding dynamics between these proteins. G Flow cytometry diagrams of CD8 + T cells for (Ga) control group, (Gb) AA group, (Gc) blebbistatin group, and (Gd) blebbistatin + AA group with statistical results ( H ) ( n = 3 for each group. ** p < 0.01, * p < 0.05). I Flow cytometry diagrams of CD11b + cells for (Ia) control group, (Ib) AA group, (Ic) blebbistatin group, and (Id) blebbistatin + AA group with statistical results ( J ) ( n = 3 for each group. **** p < 0.0001, *** p < 0.001, ** p < 0.01)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Binding Assay, Mass Spectrometry, Concentration Assay, Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Anti-CD8α antibody injection reduced the cognitive level of young mice. A Flow cytometry analysis of CD8 + T cells versus CD4 + T cells in the peripheral blood of mice after injection of IgG2b antibody and Anti-CD8α antibody ( B ). C Statistical histogram of CD8 + T flow cytometry results. D CD4 + T flow cytometry results statistical histogram. E Flow cytometry analysis of NK cells in mouse peripheral blood after IgG2b and Anti-CD8α antibody injection. F Statistical histogram of flow cytometry results of FNK cells. G Tracking diagram of mouse movement trajectory in open field test after antibody injection. H Time (s), speed, and distance traveled by the mouse in the central region during the open field test. I Statistical chart of new object cognition index ( n = 5 for each group, ns P > 0.05, * p < 0.05, *** P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Injection, Flow Cytometry

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: bioRxiv

Article Title: Anti-inflammatory effects of WFS1 in pancreatic β-cells

doi: 10.1101/2022.02.09.479773

Figure Lengend Snippet: (A) Iba1 DAB staining of the islets from Wfs1 wild type (WT) and whole-body knockout 129S6 (KO) male mice at 10 months of age. Yellow arrowheads point to the stained macrophages. Scale bars 50 µm. (B) The number of Iba1 positive cell was normalized to each islet’s area size (WT, n=21; KO, n=28). (C) Left panel: Visualized imaging mass cytometry (IMC) image of KO male mouse stained with F4/80 (red), insulin (green), DNA (blue), CD68 (magenta), and CD163 (yellow). Contrast staining was performed with Ruthenium (cyan). Middle panel: cells were masked, and the islet area was gated. Right panel: Scatter plot of F4/80, CD163, and CD68. IL-1β was shown in the heatmap. (D) Trichrome-Masson staining of the islets from WT and KO male mice at 10 months of age. Collagen fibers are stained in blue. Scale bars 50 µm. (E) The blue-colored area was normalized to each islet’s area size (WT, n=33; KO, n=34). Data are shown in mean ± SEM, * P<0.05, **** P<0.001.

Article Snippet: Blocking was performed in 2% bovine serum albumin (BSA) for 1 h. The following primary antibodies were diluted in 0.2% BSA and incubated overnight at 4 °C: anti-WFS1 (Proteintech, Cat# 1158-1-AP, 1:100), anti-Iba1 (Novus, Cat# NB100-1028, 1:50), anti-CD31 (abcam, Cat# ab124432, 1:100), and Alexa-Fluor 488 conjugated anti-insulin (Invitrogen, Cat# 53-9769-82, 1:100).

Techniques: Staining, Knock-Out, Imaging, Mass Cytometry

Journal: Cell Death & Disease

Article Title: Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

doi: 10.1038/cddis.2010.36

Figure Lengend Snippet: Cofilin translocates to the mitochondria upon oxidative stress. ( a ) Primary human T cells were incubated with (lower panel) or without (upper panel) 50 μ M H 2 O 2 . Thereafter, cells were stained for cofilin (red) or mitochondria (MitoTracker, green) and analyzed via confocal laser scan microscopy. Merge displays the digital overlay of red and green fluorescence. The figure is representative of three independent experiments. ( b ) For cryo-immunogold electron microscopy, primary human T cells were either left untreated (i and iii) or treated with H 2 O 2 (ii and iv) and subsequently fixed with 2% PFA for 10 min. Cells were stained with cofilin antiserum combined with protein A labeled with 15 nm gold particles. Shown are two example pictures taken from two independent experiments (M, mitochondria; N, nucleus). ( c , d ) The colocalization of cofilin and mitochondria was evaluated by the calculation of a similarity score of the two probes from untreated (gray histogram) and H 2 O 2 -treated (black lined histogram) PBT using MIFC. The histogram shows the distribution of the similarity within the whole-cell population as in conventional flow cytometry (up to 10 000 cells). A score of 1 indicates that the two probes are uncorrelated, whereas higher numbers indicate a higher degree of similarity. The mean similarity score of four independent experiments is shown in ( d ) ( n =4; S.E.M.; * P <0.05)

Article Snippet: The mouse monoclonal anti-FLAG antibody (clone M2, 0.2 μ g/ml) and cytochalasin D were from Sigma-Aldrich (Taufkirchen, Germany); cofilin antibodies were produced in our laboratory; HSC70 antiserum was from Santa Cruz (Heidelberg, Germany); the CD95 antibody was a kind gift of PH Krammer (DKFZ, Heidelberg, Germany); Z-VAD-fmk was from Promega (Mannheim, Germany); 7-AAD, Annexin V and anti-active caspase-3 were from BD Bioscience (Heidelberg, Germany); Hoechst 33342, MitoTracker Deep Red FM and DilC 1 (5), as well as TMRE were obtained from Invitrogen (Karlsruhe, Germany); and PJ-34 was purchased from Sigma.

Techniques: Incubation, Staining, Microscopy, Fluorescence, Electron Microscopy, Labeling, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

doi: 10.1038/cddis.2010.36

Figure Lengend Snippet: Cofilin binds to HSC70 under oxidative stress conditions. ( a ) FLAG-tagged wt- or G39-cofilin was expressed in and then precipitated from Jurkat T cells. Lysates were subjected to PAGE and stained with Coomassie blue. The heavy (50 kDa) and light (ca. 25 kDa) chains of the precipitating antibody were found in each lane, including the control (untransfected Jurkat T cells). Bands that were found only in lane 2 (G39-cofilin) or lane 3 (wt-cofilin) were analyzed using mass spectrometry and identified as HSC70 (only present in lane 2) or actin (lanes 2 and 3). ( b ) Wt-, G39- or G80-cofilin was precipitated from Jurkat T cells. The corresponding western blot was stained for the FLAG-tag (lower part) or HSC70 (upper part). ( c , d ) PBTs were treated with 50 μ M H 2 O 2 for 7 or 24 h. Thereafter, cells were lysed and endogenous cofilin was immunoprecipitated. The precipitates were subjected to western blot analysis and stained for HSC70 (upper panel) or cofilin (lower panel). The graph in ( d ) shows a quantification of the HSC70 to cofilin ratios of three independent experiments ( n =3; S.E.M.; * P <0.05). ( e ) The subcellular localization of HSC70 in long-term oxidatively stressed (lower part) or control T cells (upper part) was analyzed using confocal laser scan microscopy. The white color in the merge of the stressed cells shows the colocalization of cofilin and HSC70 with the mitochondria. The figure is representative of three experiments. ( f ) The colocalization of cofilin and mitochondria (upper graph), cofilin and HSC70 (lower graph) or HSC70 and mitochondria (central graph) was evaluated by the calculation of a similarity score of the corresponding probes ( n =3; S.E.M.; * P <0.05)

Article Snippet: The mouse monoclonal anti-FLAG antibody (clone M2, 0.2 μ g/ml) and cytochalasin D were from Sigma-Aldrich (Taufkirchen, Germany); cofilin antibodies were produced in our laboratory; HSC70 antiserum was from Santa Cruz (Heidelberg, Germany); the CD95 antibody was a kind gift of PH Krammer (DKFZ, Heidelberg, Germany); Z-VAD-fmk was from Promega (Mannheim, Germany); 7-AAD, Annexin V and anti-active caspase-3 were from BD Bioscience (Heidelberg, Germany); Hoechst 33342, MitoTracker Deep Red FM and DilC 1 (5), as well as TMRE were obtained from Invitrogen (Karlsruhe, Germany); and PJ-34 was purchased from Sigma.

Techniques: Staining, Control, Mass Spectrometry, Western Blot, FLAG-tag, Immunoprecipitation, Microscopy

Journal: Journal of Innate Immunity

Article Title: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

doi: 10.1159/000499342

Figure Lengend Snippet: Urinary exosome lactoferrin is significantly increased during UPEC infection. a Proportion of proteins or protein families detected in urinary exosomes of UPEC-infected WT C57Bl/6 mice 24 h postinfection with 2–3 × 107 CFU CFT073 or uninfected controls. Data represent the combined results of 4 independent experiments and LC-MS/MS runs (6 independent pooled urine samples, n> 10/group). Normalized spectral abundance factor (NSAF) of lactoferrin (b), myeloperoxidase (c), and neutrophil elastase (d) from the same LC-MS/MS experiments as in a. Symbols represent 6 independent pooled biological replicates/group with lines indicating median and 95% CI. b–d Data were analyzed with the Wilcoxon matched-pairs signed rank test. * p < 0.05.

Article Snippet: To probe for lactoferrin in HTB-9 exosomes, the following antibodies were used: goat anti-lactoferrin antibody at 1:500 (Santa Cruz Biotechnology, Cat# sc-14434) and anti-β-actin monoclonal antibody at 1:2,000 (Clone AC-74, Sigma Aldrich, Cat# A5316).

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Innate Immunity

Article Title: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

doi: 10.1159/000499342

Figure Lengend Snippet: Bladder epithelial cells produce lactoferrin in response to UPEC infection. a Western blot of exosomes from HTB-9 cells infected with CFT073 for 2 h, MOI of 20, and probed for human lactoferrin (approx. 80 kDa, top image) and β-actin (bottom image) as a loading control. A single representative blot from 3 independent experiments is shown. b Normalized lactoferrin mRNA from HTB-9 cells infected with CFT073 for 2 h, MOI of 0.1, as quantified by qPCR. Symbols represent 6–7 independent replicates/group, with lines indicating mean ± SEM. c Lactoferrin production in HTB-9 cell lysate infected with CFT073, MOI of 0.1, or treated with deferoxamine (DFO, 300 µM) for 4 h as quantified by ELISA. Symbols represent means of independent experiments (n = 4–5) with lines indicating mean ± SEM. d Percent adherence of CFT073 to HTB-9 cells after 30 min of infection, MOI = 0.1. Symbols represent means of independent experiments (n = 4/group) with lines indicating mean ± SEM. Data were analyzed using the two-tailed Mann-Whitney U test (b), or one-way ANOVA with the Holm-Sidak multiple comparisons test (c, d). ** p < 0.01. hLf, human lactoferrin; Fe-Lf, iron-saturated lactoferrin.

Article Snippet: To probe for lactoferrin in HTB-9 exosomes, the following antibodies were used: goat anti-lactoferrin antibody at 1:500 (Santa Cruz Biotechnology, Cat# sc-14434) and anti-β-actin monoclonal antibody at 1:2,000 (Clone AC-74, Sigma Aldrich, Cat# A5316).

Techniques: Infection, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Innate Immunity

Article Title: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

doi: 10.1159/000499342

Figure Lengend Snippet: Human lactoferrin is not antimicrobial in synthetic urine. Growth curves of CFT073 in synthetic urine (a) or RPMI-1640 (b) with given concentrations of hLf and Fe-Lf as measured by optical density (OD). c Viable CFU/mL in RPMI-1640 were measured by serial dilution and plating. Symbols represent the means of independent experiments (n = 3–4/group) with lines indicating mean ± SEM. All data were analyzed using two-way ANOVA with the Tukey multiple comparisons test. * p < 0.05.

Article Snippet: To probe for lactoferrin in HTB-9 exosomes, the following antibodies were used: goat anti-lactoferrin antibody at 1:500 (Santa Cruz Biotechnology, Cat# sc-14434) and anti-β-actin monoclonal antibody at 1:2,000 (Clone AC-74, Sigma Aldrich, Cat# A5316).

Techniques: Serial Dilution

Journal: Journal of Innate Immunity

Article Title: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

doi: 10.1159/000499342

Figure Lengend Snippet: Exogenous lactoferrin alters neutrophil antimicrobial functions. Isolated human neutrophils were pretreated with 250 µg/mL hLf or Fe-Lf as indicated in Methods. a Neutrophil killing of CFT073 expressed as a percentage of inoculum and normalized to mock-treated controls within biological replicates. b Neutrophil extracellular trap (NET) formation by production by unstimulated neutrophils (control), or neutrophils stimulated with PMA as measured by fluorescence. c Reactive oxygen species (ROS) production by unstimulated neutrophils (control) or neutrophils stimulated with PMA and normalized to maximum values of mock-treated stimulated neutrophils within biological replicates. Symbols represent the means of biological replicates (n =3–7/group), with lines indicating median and 95% CI. Data were analyzed using the Kruskal-Wallis test with the Dunn multiple comparisons test (a), or two-way ANOVA with the Sidak multiple comparisons test (b, c). *** p < 0.01, ** p < 0.01, * p < 0.05 compared to mock-treated cells within the group (i.e., control or stimulated). hLf, human lactoferrin; Fe-Lf, iron-saturated lactoferrin.

Article Snippet: To probe for lactoferrin in HTB-9 exosomes, the following antibodies were used: goat anti-lactoferrin antibody at 1:500 (Santa Cruz Biotechnology, Cat# sc-14434) and anti-β-actin monoclonal antibody at 1:2,000 (Clone AC-74, Sigma Aldrich, Cat# A5316).

Techniques: Isolation, Control, Fluorescence

Journal: Journal of Innate Immunity

Article Title: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

doi: 10.1159/000499342

Figure Lengend Snippet: Exogenous lactoferrin protects against UPEC UTI in vivo. a Bone marrow-derived murine neutrophils were pretreated with 250 µg/mL hLf and killing of CFT073 was expressed as a percentage of inoculum. Symbols represent the means of biological replicates (n =3/group), with lines indicating median and 95% CI. WT C57Bl/6 mice were transurethrally treated with hLf 1 h prior to infection with 2–3 × 107 CFU CFT073, or mock-treated as a control. b Bladder and kidney bacterial burdens quantified 24 h postinfection. c Ly6G+/CD11b+ and Ly6G–/CD11b+ cells in mouse urine collected 24 h postinfection and quantified via flow cytometry. Symbols represent biological replicates (n = 7–17/group), with lines indicating median and 95% CI. Data were analyzed using the two-tailed paired Student t test (a) or two-way ANOVA with the Sidak multiple comparisons test (b, c). *** p < 0.01, * p < 0.05.

Article Snippet: To probe for lactoferrin in HTB-9 exosomes, the following antibodies were used: goat anti-lactoferrin antibody at 1:500 (Santa Cruz Biotechnology, Cat# sc-14434) and anti-β-actin monoclonal antibody at 1:2,000 (Clone AC-74, Sigma Aldrich, Cat# A5316).

Techniques: In Vivo, Derivative Assay, Infection, Control, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: DTX3L and USP28 interact with each other (A) Interaction of endogenous DTX3L and USP28 was visualized by proximity ligation assay (PLA) in SK-MES-1 cells. Cells were grown on cover slips, fixed and immunostained according to Duolink manufacture’s protocol. Red spots reflect the interaction. DAPI stained nuclei are shown in blue. Scale bars: A-A‴ 50 μm and a-a’’’ 20 μm. (B) Quantification of PLA signals. ∗∗∗∗significant difference between the number of proximity ligation sites in the negative control vs. DTX3L/USP28. Data are mean ± SD from 3 independent experiments. The statistical significance of differences was determined by ordinary one-way ANOVA. ∗∗∗ p < 0.001. (C and D) Cell extracts from PC-3 cells were immunoprecipitated with either IgG control or USP28 antibodies. Resulting precipitates were first analyzed by immunoblotting (IB) using a DTX3L; thereafter, the membrane was re-probed with a USP28 antibody. Whole cell lysate (C) and cytoplasmic (CF) and nuclear fraction (NF) (D). (E) HEK293 cells were cotransfected with expression vectors encoding full length Flag-tagged USP28 and full-length HA-tagged DTX3L or the N-terminal HA-tagged DTX3L (A2-A516) deletion mutant or the C-terminal HA-tagged DTX3L (K555-E740) deletion mutant. Cell extracts were immunoprecipitated with either IgG control or USP28 antibodies. Immunoprecipitates were analyzed by immunoblotting (IB) using an HA-tag antibody. (F) Representative MST binding curve for DTX3L and USP28 interaction. All binding curves are shown in .

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Proximity Ligation Assay, Staining, Ligation, Negative Control, Immunoprecipitation, Control, Western Blot, Membrane, Expressing, Mutagenesis, Binding Assay

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Functional interplay between USP28 and DTX3L (A) In vitro ubiquitination assay indicating the auto-ubiquitination activity of DTX3L and deubiquitinating activity of USP28. Protein concentrations used were Ub (50 μM), E1 (0.4 μM), UbcH5a (E2) (2 μM) and DTX3L (0.7 μM). USP28 was used at a final concentration of 3 μM. SDS-PAGE showing appearance of the DTXL3 ubiquitination pattern that is visible as a smear (lane 8) and its disappearance due to hydrolysis by USP28 (lane 9) and/or the USP28 catalytic domain (lane 11). Ubiquitinated DTX3L appears as a high molecular weight smear and the black arrow indicates a band that corresponds to ubiquitinated USP28. Note, that for unknown reasons USP28 preparations showed some degradation (lanes 5–6). (B) Western blot of the assay in panel A probed with an anti-ubiquitin antibody. The black arrow indicates a band that corresponds to ubiquitinated USP28. (C) Western blot of the assay in panel A probed with anti-USP28. The arrow indicates a band shift of the ubiquitinated catalytically inactive mutant USP28 C171A in the presence of ATP, Ubiquitin (Ub), E1 and E2 ligases as well as DTX3L. (D–H) Cell based ubiquitination assays. HEK293 cells were transfected with an empty vector (Ctl) or expression vectors for DTX3L, wild type USP28 and the catalytically inactive mutant USP28 C171A . In D, ubiquitinated DTX3L was detected with ubiquitin antibody after immunoprecipitation of DTX3L. In (E), ubiquitinated USP28 was detected with ubiquitin antibody after immunoprecipitation of USP28 with a FlagM2-tag antibody. In (F), cells were transfected with DTX3L and Flag-USP28 expression vectors as well as with empty vector (Ctl) or vectors for wild type ubiquitin (Ub), a mutant in which all lysine residues were converted to arginine (Ub-KR) or mutants where either only K48 (Ub-K48) or K63 (Ub-K63) remained intact. Ubiquitinated USP28 was detected with ubiquitin antibody after immunoprecipitation of USP28 with a FlagM2-tag antibody. In (H), cells were transfected with HA-DTX3L and Flag-USP28 expression vectors as well as with empty vector (Ctl) or vectors for wild type ubiquitin and the different ubiquitin mutants as in (F). Ubiquitinated DTX3L was detected with ubiquitin antibody after immunoprecipitation of DTX3L with an HA-tag antibody. (G) Domain organization of USP28 and the ubiquitination sites identified by mass spectrometry using the USP28 C171A mutant. UBA - ubiquitin-associated domain; UIM -ubiquitin interaction motif; SIM - SUMO interaction motif.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Functional Assay, In Vitro, Ubiquitin Proteomics, Activity Assay, Concentration Assay, SDS Page, High Molecular Weight, Western Blot, Electrophoretic Mobility Shift Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Mass Spectrometry

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Mutual regulation of DTX3L and USP28 protein levels (A) SK-MES-1 cells were transfected either with expression vector for scrambled control shRNA (shScr), or with expression vectors for full-length DTX3L or one of two independent shRNAs (shDTX3L_8, shDTX3L_11) against DTX3L. After transfection, cells were further cultured under normoxia (16% O 2 ) or hypoxia (5% O 2 ) for 4 h. (B) USP28, HIF-1α, p53, c-MYC, and DTX3L protein levels were measured by Western blot analysis. Alpha tubulin served as a loading control. (C) SK-MES-1 cells were treated with the USP28/25 inhibitor AZ1 (10 μM) and cultured under normoxia and hypoxia for 4 h and 24 h. (D) DTX3L, HIF-1α, p53, and c-MYC protein levels were measured by Western blot analysis. (E) SK-MES-1 cells were transfected with expression vectors for scrambled control shRNA (shScr) or shRNA 1 or shRNA 3 against USP28. After transfection, cells were further cultured under normoxia or hypoxia for 4 h. DTX3L, HIF-1α, p53, c-Myc and USP28 protein levels were measured by Western blot analysis. (B–F) Quantification of USP28, DTX3L, HIF-1α, p53 and c-MYC. In each experiment the protein levels at 5% O 2 control (Ctl) or shScr were set to 100%. ∗significant difference for 16% O 2 : Ctl vs. DTX3L, vs. shDTX3L, vs. shUSP28 or vs. AZ1, § significant difference between 16% O 2 vs. 5% O 2 , # significant difference for 5% O 2 : Ctl vs. DTX3L, vs. shDTX3L, vs. shUSP28 or vs. AZ1, p < 0.05.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, shRNA, Cell Culture, Western Blot

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: DTX3L and USP28 control each other’s half-life (A and C) HEK293 cells were transfected with scrambled shRNA or with two different shRNA’s against either DTX3L or USP28. After transfection, protein synthesis was inhibited with cycloheximide (CHX; 10 μg/μL) and cells were harvested at indicated time points. In each experiment the protein levels at time point 0 were set to 100%. ∗significant differences shScr vs. shDTX3L or shUSP28_1, # significant differences shScr vs. shDTX3L_11 or shUSP28_3, p ≤ 0.05. (B and D) Representative Western blot analysis. 100 μg of total protein lysate was analyzed with antibodies against USP28, DTX3L and α-tubulin.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Control, Transfection, shRNA, Western Blot

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: NHEJ-, and HR-DSB repair analysis (A) Induction of DSBs by bleomycin (BLM) does not affect USP28/DTX3L interaction. Cells treated with BLM (10 μM) and the proteasome inhibitor MG132 were immunoprecipitated with either IgG control or USP28 antibodies. Resulting precipitates were analyzed by immunoblotting (IB) using a DTX3L antibody. Membrane was re-probed with an antibody against USP28. (B and E) Scheme of pathway-specific repair activities for NHEJ and HR measured with EGFP reporter assays. (C and F). Quantification of NHEJ and HR DSB repair activities in BLM treated MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells. Mean values for the MDA-MB-231 Scr cells (shScr) were defined as 100%. Data are mean ± SD from 6 measurements. The statistical significance of differences was determined using ordinary one-way ANOVA. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (D and G) Representative flow cytometry images.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Immunoprecipitation, Control, Western Blot, Membrane, Knockdown, Flow Cytometry

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: SSA-, and MMEJ-DSB repair analysis (A and D) Scheme of pathway-specific repair activities for NHEJ and HR measured with EGFP reporter assays. (B and E). Quantification of NHEJ and HR DSB repair activities in BLM treated MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells. Mean values for the MDA-MB-231 Scr cells (shScr) were defined as 100%. Data are mean ± SD from 6 measurements. The statistical significance of differences was determined using ordinary one-way ANOVA. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C and F) Representative flow cytometry images. (G) Representative images of Comet assay. Scale bar: 1000 μM. (H) Determination of % of DNA in tail in MDA-MB-231 cells transfected with the indicated shRNAs. Data were expressed as mean ± SD of three independent experiments ( n > 50 cells per condition). Statistical significance was determined using ordinary one-way ANOVA. ∗∗∗∗ p < 0.001. (I) Representative immunofluorescence images of γH2AX (green) and HOECHST nuclear staining (blue). Scale bar:50 μm. (J) Quantification of mean γH2AX foci per nucleus in MDA-MB-231 cells transfected with the indicated shRNAs. Data are mean ± SD from three experiments ( n > 100 cells per condition). Statistical significance was determined using ordinary one-way ANOVA. ∗∗∗∗ p < 0.001.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Knockdown, Flow Cytometry, Single Cell Gel Electrophoresis, Transfection, Immunofluorescence, Staining

Journal: iScience

Article Title: The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels

doi: 10.1016/j.isci.2025.112990

Figure Lengend Snippet: Mutual regulation of cell cycle and apoptosis by USP28 and DTX3L upon DNA DSB induction. MDA-MB-231 Scr, USP28-KD, DTX3L-KD and USP28/DTX3L double knockdown cells were treated with bleomycin (BLM; 10 μM) and further cultured for 24 h (A) Cell cycle distribution of MDA-MB-231 shScr, USP28 KD, DTX3L KD, and double knockdown (USP28 KD + DTX3L KD) cells in the G1/0 phase, S phase, and G2 phase. Significance ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (B) Quantification of apoptosis in MDA-MB-231 Scr, USP28 KD, DTX3L KD and USP28/DTX3L double knockdown cells Significance ∗ p < 0.05, ∗∗ p < 0.01. (C) Histograms of apoptotic cells assessed by Annexin-V/PI staining and measured by flow cytometry.

Article Snippet: Blocking was performed in PBS containing 5% fetal bovine and 5% goat serum for 1 h. This was followed by incubation with primary antibodies against γH2X (1:500, #05–636, Millipore), DTX3L (1:100, #sc-514776, Santa Cruz) and USP28 (1:100; #HPA006778; Sigma-Aldrich); both were diluted in 1% blocking buffer and incubated with the coverslips overnight at 4°C.

Techniques: Knockdown, Cell Culture, Staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A ) Senescence was induced in primary human endothelial cells (HUVECs) by repeated subculture over 35 passages or treatment with doxorubicin. In doxycycline-inducible immortalized human endothelial cells (HuARLT cells), senescence was induced by culturing without doxycycline. SA-β-gal staining was used for validation of senescence through microscopy or flow cytometry. p, cell culture passages. dc, doxycycline. KSHV infectivity was measured by GFP expression in cells infected with recombinant KSHV BAC16. ( B ) Analysis of KSHV-infected cells in nonsenescent and senescent human endothelial cells by flow cytometry at 24 hpi. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. *** P < 0.001, unpaired 2-tailed Student’s t test. ( C ) Quantification of the KSHV genome in KSHV-infected nonsenescent and senescent human endothelial cells by quantitative PCR at 24 hpi. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ** P < 0.01, *** P < 0.001, unpaired 2-tailed Student’s t test. ( D ) Assessment of the relative expression of KSHV ORF71 mRNA in KSHV-infected nonsenescent and senescent human endothelial cells using quantitative reverse transcription PCR at 24 hpi. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ** P < 0.01, unpaired 2-tailed Student’s t test.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Staining, Biomarker Discovery, Microscopy, Flow Cytometry, Cell Culture, Infection, Expressing, Recombinant, Real-time Polymerase Chain Reaction, Reverse Transcription

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A ) Immunofluorescence assay of the entry of KSHV into nonsenescent and senescent cells. KSHV was visualized at 4 hpi using KSHV ORF65 antibody. Phalloidin (F-actin) and DAPI were used to visualize the shapes of the cells and nuclei, respectively. Scale bars: 50 μm. ( B ) Analysis of the number of internalized KSHV particles per cell in the images in A and . Data are representative of 10 independent experiments. Data are shown as mean ± SD. n = 10. *** P < 0.001 using unpaired 2-tailed Student’s t test. ( C ) Quantification of the internalized KSHV genome in the KSHV-infected nonsenescent and senescent human endothelial cells by quantitative PCR. Genomic DNA was extracted from KSHV-infected cells at 4 hpi. The KSHV genome was quantified in the extracted DNA by quantitative PCR using primers for KSHV ORF26. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ** P < 0.01; *** P < 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Immunofluorescence, Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A ) Confocal microscopy images of KSHV binding to the cell surface. After infection, cells were immediately fixed and stained without permeabilization. Scale bars: 10 μm. ( B ) Analysis of the number of KSHV particles binding to the cell surface per cell in the images in A and . Data are representative of 10 independent experiments. Data are shown as mean ± SD. n = 10. *** P < 0.001, unpaired 2-tailed Student’s t test. ( C ) Quantification of the cell-surface–bound KSHV genome in KSHV-infected cells by quantitative PCR. After 1 hour of KSHV infection, the cells were immediately scraped and genomic DNA was extracted. The KSHV genome was quantified in the extracted DNA by quantitative PCR using primers for KSHV ORF26. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ** P < 0.01; *** P < 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Confocal Microscopy, Binding Assay, Infection, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A ) Heatmap of the differential expression of proteins from nonsenescent (+dc, culture with doxycycline) and senescent (–dc, culture without doxycycline) HuARLT cells with KSHV (K) or without KSHV (M). The cell lysate and cell pellet from each conditioned cell were analyzed by LC-MS/MS. ( B ) Western blot analysis of the selected candidate proteins. Cav-1, caveolin-1; ITG, integrin; dc, doxycycline. GAPDH was used as a housekeeping protein for normalization. ( C ) Flow cytometric analysis of candidate proteins in nonsenescent (+dc HuARLT and –dox HUVEC) and senescent (–dc HuARLT and +dox HUVEC) cells. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. * P < 0.05; ** P < 0.01; *** P < 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: An equivalent quantity of KSHV was used to infect the same number of WT and KO HuARLT cells, with or without induction of senescence. KSHV was prepared as GFP infectious units of 1 to infect approximately 90% of nonsenescent WT cells, followed by the infection of a 2-fold serially diluted virus into each group of conditioned cells. KSHV infectivity was measured using GFP expression. ( A and B ) Fluorescence microscopic visualization of KSHV-infected cells for each KO clone in nonsenescent (+dc, A ) and senescent (–dc, B ) HuARLT cells. CAV1 , caveolin-1; ITGA2 , integrin-α 2 . Scale bar: 100 μm. ( C and D ) Flow cytometric analysis of KSHV-infected cells for each KO clone in nonsenescent (+dc, C ) and senescent (–dc, D ) HuARLT cells. At the indicated GFP infectious units (GIU), the percentage of KSHV-infected cells was compared among each group. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. *** P < 0.001, Dunnett’s test for multiple comparisons.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Infection, Virus, Expressing, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A ) Representative confocal images of KSHV virus particles in CAV1 -KO and CD109 -KO HuARLT cells. Scale bars: 10 μm. ( B ) Analysis of the number of KSHV particles that bind to the cell surface per cell in the images in A and . Data are representative of 10 independent experiments. Data are shown as mean ± SD. n = 10. *** P < 0.001, Dunnett’s test for multiple comparisons. ( C ) Quantification of the cell-surface–bound KSHV genome in the KSHV-infected cells by quantitative PCR. After 1 hour of KSHV infection, the cells were immediately scraped, and genomic DNA extracted after washing. The KSHV genome was quantified in the extracted DNA by quantitative PCR using primers for KSHV ORF26. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ** P < 0.01; *** P < 0.001, Dunnett’s test for multiple comparisons.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Virus, Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: ( A and B ) Three-dimensional confocal microscopy images of the colocalization of KSHV with CD109 ( A ) or caveolin-1 (Cav-1) ( B ). Senescent HuARLT cells infected with KSHV for 1 hour were stained with KSHV K8.1 antibody and target proteins, and Z -axis scanning was performed at 4 μm intervals, generating more than 10 scans. The bar-shaped images at the edges represent cross-sections, guided by white solid lines in the stacked image. ( C ) Quantitative analysis of the colocalization of KSHV with CD109 or caveolin-1 by Manders’ colocalization coefficient in KSHV-infected senescent HuARLT cells. n = 10. *** P < 0.001 ( D ) SPR analysis of KSHV binding to immobilized recombinant CD109. Data are representative of 3 independent experiments. ( E ) Neutralization of KSHV infectivity using a recombinant CD109 protein. Data are representative of 3 independent experiments. Data are presented as mean ± SD. n = 3. *** P < 0.001, Dunnett’s test for multiple comparisons.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Confocal Microscopy, Infection, Staining, Binding Assay, Recombinant, Neutralization

Journal: The Journal of Clinical Investigation

Article Title: Senescence of endothelial cells increases susceptibility to Kaposi’s sarcoma–associated herpesvirus infection via CD109-mediated viral entry

doi: 10.1172/JCI183561

Figure Lengend Snippet: Co-IP analysis of CD109 with KSHV glycoproteins gB ( A ), gH/gL ( B ), and K8.1 ( C ). HEK-293T cells were cotransfected with the indicated plasmids for 24 hours, after which cell lysates were subjected to IP using anti-FLAG magnetic agarose beads. The resulting complexes were then analyzed by immunoblotting (IB) using the indicated antibodies. Data are representative of 3 independent experiments.

Article Snippet: Mouse anti-KSHV K8.1 antibody (1:100 dilution; SC-65446; 4A4; Santa Cruz Biotechnology), rabbit anti-caveolin-1 (1:100 dilution; A1555; Abclonal), rabbit anti-CD109 (1:100 dilution; 486955; E8L2W; Cell Signaling Technology), rat anti-LANA (1:100 dilution; Ab4103; LN53; Abcam), and mouse anti-KSHV ORF 65 (1:100 dilution) ( ) were used as primary antibodies.

Techniques: Co-Immunoprecipitation Assay, Western Blot