Journal: eLife

Article Title: Apoptotic signaling clears engineered Salmonella in an organ-specific manner

doi: 10.7554/eLife.89210

Figure Lengend Snippet: ( A ) Schematic of engineered BID ON construct. ( B ) Pathway model showing how BID ON leads to intrinsic apoptosis. ( C–G ) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. Typhimurium strains. ( C–E ) Western blot analysis of whole cell lysates at 6 hpi. Data representative of two ( C ) or three ( D–E ) independent experiments. ( F ) Western blot analysis of cytosolic and mitochondrial fractions at 4 hpi. Data representative from three independent experiments. ( G ) Brightfield at 6 hpi. Data representative of three independent experiments. 60 x magnification, scale bar 20 µm, carrot, apoptotic blebs. ( H ) Z-stack slices from . WT BMMs infected with BID ON imaged at 6 hpi. Representative Z-stack from three (brightfield, PI) or one (cleaved caspase-3/7) independent experiments. 60 x magnification, Z-slices 11, 19, 22, and 24 shown. Carrots; selected examples of apoptotic bodies. Figure 3—source data 1. Western blot images for . Figure 3—source data 2. Western blot images for . Figure 3—source data 3. Western blot images for . Figure 3—source data 4. Western blot images for .

Article Snippet: Protein was then transferred onto a 0.45 μm PVDF membrane (Millipore, Burlington, MA, Cat. IPFL85R), blocked with 5% non-fat dried milk in TBS plus 0.01% Tween (TBST) for 1 hr at room temperature, and incubated overnight at 4 °C with mild agitation in 5% milk in TBST plus indicated antibody: cytochrome c (1:750, rabbit, Cell Signaling Technology, Danvers, MA, Cat. 11940), GAPDH (1:10,000, rabbit, abcam, Cambridge, UK, Cat. Ab9485), VDAC (1:750, rabbit, Cell Signaling Technology, Cat. 4661), cleaved caspase-8 (1:1000, rabbit, Cell Signaling Technology, Cat. 8592), BID (1:500, rat, R&D Systems, Minneapolis, MN, Cat. MAB860), caspase-9 (1:750, mouse, Cell Signaling Technology, Cat. 9508), cleaved caspase-7 (1:1000, rabbit, Cell Signaling Technology, Cat. 9491), cleaved caspase-3 (1:750, rabbit, Cell Signaling Technology, Cat. 9661), gasdermin D (1:1000, rabbit, abcam, Cat. ab209845), or HA (1:2000, mouse, Biolegend, San Diego, CA, Cat. MMS-101R).

Techniques: Construct, Derivative Assay, Infection, Western Blot

Journal: eLife

Article Title: Apoptotic signaling clears engineered Salmonella in an organ-specific manner

doi: 10.7554/eLife.89210

Figure Lengend Snippet: ( A–B ) Bone marrow-derived macrophages (BMMs) were infected with indicated SPI2-induced S. Typhimurium strains. ( A ) Western blot analysis of whole cell lysates. Representative of five independent experiments. ( B ) Immunofluorescence and brightfield. Cells were stained with PI, cleaved caspase-3/7, and Hoechst and imaged at indicated timepoints. Representative image from three (brightfield, PI) or one (cleaved caspase-3/7) independent experiments. Z-stack of the 6 hr timepoint is represented in and . Z-stack slice 19 (FliC ON in Gsdmd –/– ) and slice 20 (BID ON in WT) shown here. 60 x magnification, scale bar 20 µm. Arrows, pyroptotic cells. Carrots, apoptotic cells. Figure 4—source data 1. Western blot images for .

Article Snippet: Protein was then transferred onto a 0.45 μm PVDF membrane (Millipore, Burlington, MA, Cat. IPFL85R), blocked with 5% non-fat dried milk in TBS plus 0.01% Tween (TBST) for 1 hr at room temperature, and incubated overnight at 4 °C with mild agitation in 5% milk in TBST plus indicated antibody: cytochrome c (1:750, rabbit, Cell Signaling Technology, Danvers, MA, Cat. 11940), GAPDH (1:10,000, rabbit, abcam, Cambridge, UK, Cat. Ab9485), VDAC (1:750, rabbit, Cell Signaling Technology, Cat. 4661), cleaved caspase-8 (1:1000, rabbit, Cell Signaling Technology, Cat. 8592), BID (1:500, rat, R&D Systems, Minneapolis, MN, Cat. MAB860), caspase-9 (1:750, mouse, Cell Signaling Technology, Cat. 9508), cleaved caspase-7 (1:1000, rabbit, Cell Signaling Technology, Cat. 9491), cleaved caspase-3 (1:750, rabbit, Cell Signaling Technology, Cat. 9661), gasdermin D (1:1000, rabbit, abcam, Cat. ab209845), or HA (1:2000, mouse, Biolegend, San Diego, CA, Cat. MMS-101R).

Techniques: Derivative Assay, Infection, Western Blot, Immunofluorescence, Staining

Journal: eLife

Article Title: Apoptotic signaling clears engineered Salmonella in an organ-specific manner

doi: 10.7554/eLife.89210

Figure Lengend Snippet:

Article Snippet: Protein was then transferred onto a 0.45 μm PVDF membrane (Millipore, Burlington, MA, Cat. IPFL85R), blocked with 5% non-fat dried milk in TBS plus 0.01% Tween (TBST) for 1 hr at room temperature, and incubated overnight at 4 °C with mild agitation in 5% milk in TBST plus indicated antibody: cytochrome c (1:750, rabbit, Cell Signaling Technology, Danvers, MA, Cat. 11940), GAPDH (1:10,000, rabbit, abcam, Cambridge, UK, Cat. Ab9485), VDAC (1:750, rabbit, Cell Signaling Technology, Cat. 4661), cleaved caspase-8 (1:1000, rabbit, Cell Signaling Technology, Cat. 8592), BID (1:500, rat, R&D Systems, Minneapolis, MN, Cat. MAB860), caspase-9 (1:750, mouse, Cell Signaling Technology, Cat. 9508), cleaved caspase-7 (1:1000, rabbit, Cell Signaling Technology, Cat. 9491), cleaved caspase-3 (1:750, rabbit, Cell Signaling Technology, Cat. 9661), gasdermin D (1:1000, rabbit, abcam, Cat. ab209845), or HA (1:2000, mouse, Biolegend, San Diego, CA, Cat. MMS-101R).

Techniques: Plasmid Preparation, Knock-Out, Produced, Western Blot, Recombinant, Lactate Dehydrogenase Assay, Software

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Quantification of internalized B19V. Lentivirus-transduced UT7/Epo-S1 cells (S1 shAXL or S1 shScr ) were incubated with B19V at an MOI of 1000 at 37°C for 1.5 hours. After treatment with trypsin and benzonase, the cells were washed with PBS three times, and viral DNA was extracted for quantification by qPCR. ( B to D ) Quantification of B19V replication and egress. S1 shAXL or S1 shScr cells were infected with B19V at an MOI of 1000. At 2 dpi, the cells were collected for quantification of B19V replication by relative viral DNA levels versus mitochondrial DNA (B), relative VP2 mRNA versus β-actin mRNA (C), or virus egress (D). The values (mean ± SD) obtained from S1 shAXL are normalized to those from the S1 shScr cells, which are arbitrarily set up to 100% [(D), S1 shScr ]. All the experiments were performed in triplicate, and the data were analyzed by Student’s t test (** P < 0.01 and **** P < 0.0001).

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Infection, Virus

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: Lentivirus-transduced CD36 + EPCs were incubated with B19V at an MOI of 1000 at 37°C for 1.5 hours. ( A ) Quantification of internalized B19V. Cells were treated with trypsin and benzonase, followed by washing with PBS. The internalized B19V was quantified by qPCR. ( B and C ) Quantification of B19V replication. At 2 dpi, the indicated cells were collected and treated with benzonase, followed by quantification of B19V DNA using qPCR (B) and VP2 mRNA using RT-qPCR (C). ( D ) Immunofluorescence assay. At 2 dpi, cells were collected and resuspended in DMEM and cytospun onto slides, followed by fixation, permeabilization, and sequentially staining with anti-B19V capsid (clone 521-5D) and an FITC-conjugated secondary antibody (green). Confocal images were taken at a magnification of ×20. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E and F ) Flow cytometry. At 2 dpi, infected EPCs were fixed, permeabilized, and stained with anti-B19V capsid (#clone 521-5D) and an FITC-conjugated secondary antibody, sequentially, followed by flow cytometry (E). All the experiments were performed in triplicate (F), and the data shown were analyzed by Student’s t test (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Quantitative RT-PCR, Immunofluorescence, Staining, Flow Cytometry, Infection

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A and D ) Quantification of B19V DNA replication. (A) CD36 + EPCs were incubated with an anti-AXL or an IgG isotype control (at indicated concentrations) at 4°C before B19V infection (MOI = 1000). (D) CD36 + EPCs were incubated with soluble rAXL EC or MBP (0.1 to 10 μg/ml) during B19V infection. At 2 dpi, the cells were collected and quantified for replicated B19V genome and mitochondrial DNA using qPCR. The number of replicated B19V genomes of each group is standardized with the mitochondrial DNA numbers. Data shown are relative to the mock-treated cell group, which is arbitrarily set up as 100%. ( B and C ) Immunofluorescence assay and flow cytometry. CD36 + EPCs were incubated with anti-AXL or IgG isotype control (10 μg/ml) at 4°C before B19V infection. At 2 dpi, the cells were collected and stained with an anti-capsid antibody (clone 521-5D), followed by immunofluorescence assay (B) and flow cytometry (C), respectively. Scale bar, 50 μm.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Control, Infection, Immunofluorescence, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Immunofluorescent assay for colocalization of AXL with B19V virions. CD36 + EPCs cells were infected with B19V at an MOI of ~5000 vgc per cell for 1.5 hours at 4°C. The cells were then washed with ice-cold PBS, then cytospun onto slides, fixed, permeabilized, and stained with an anti-B19V capsid antibody (clone MAB860-55D). Confocal images were taken under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. ( B to D ) Kinetics of the in vitro interaction between AXL and VP1u using Ni-NTA biosensors. The binding kinetics depicts the associations and dissociations of 4 μM rAXL EC with GST-VP1u at the indicated concentrations (B). Binding kinetics of 4 μM AXL His with 4 and 8 μM GST was used as a control (C). The binding parameters of AXL and GST-VP1u are shown (D). Equilibrium dissociation constant K D value represents the ratio of dissociation [ K d (1/s)] and association [ K a (1/Ms)] computed from the real-time binding curves of rAXL EC to GST-VP1u. The values are shown with means ± SDs based on at least three repeated experiments.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Infection, Staining, Microscopy, In Vitro, Binding Assay, Control

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Cell surface expression of AXL. K562 cells were transduced with lentivirus-expressing AXL protein to express AXL on the cell surface (K562 AXL ). Blank lentivirus vector serves as control (K562 lentictrl ). Flow cytometry was used to analyze cell surface expression of AXL. ( B and C ) Overexpression of AXL on K562 cells increases B19V entry and infection. K562, K562 AXL , and K562 lentictrl cells were infected with B19V at an MOI of ~2000 at 37°C for 1.5 hours. (B) Internalized B19V virions were quantified by qPCR. (C) At 2 dpi, total DNA was extracted and analyzed for the replicated viral DNA and mitochondrial DNA by qPCR. The numbers shown are mitochondrial DNA–standardized B19V genome levels of the infected K562 AXL and K562 lentictrl cells related to that of the control K562 cells.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Flow Cytometry, Over Expression, Infection