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