(A) Schematic representation of Casp11 fluorescent reporter constructs used in this study indicating mCherry fused to the C-terminus of wild-type (WT), catalytically inactive (C254A), or …
Source data for Figure 1B.
Caspase-11-mediated membrane pore formation was assessed by imaging primary bone marrow-derived macrophages (BMDMs) after uptake of Live/Dead green fluorescent dye. Nuclei are stained with Hoechst.
Source data for Figure 1C.
Casp11-mCherry expression and gasdermin D (GSDMD) processing in response to lipopolysaccharide (LPS) transfection was assessed by western blotting for mCherry and GSDMD as indicated. β-actin was used as a loading control.
Source data for Figure 1D.
Bone marrow-derived macrophages (BMDMs) expressing indicated Casp11-mCherry constructs were fixed post-lipopolysaccharide (LPS) transfection and prepared for confocal microscopy. Nuclei are stained with Hoechst.
Source data for Figure 1F.
Bone marrow-derived macrophages (BMDMs) expressing indicated Casp11-mCherry constructs were fixed post-infection with Legionella pneumophila (MOI = 50) and prepared for confocal microscopy. Nuclei are stained with Hoechst.
(A) Wild-type (WT), catalytically inactive (C254A), and non-cleavable (D285A) Casp11-mCherry expression plasmids were transfected (0.25 μg) into HEK293T cells. Cell lysates were immunoblotted for …
Source data for Figure 2A.
Indicated Casp11-mCherry expression plasmids were transfected into HEK293T cells. Cell lysates were immunoblotted for mCherry, Casp11, and β-actin (loading control) 10 hr post-transfection.
Source data for Figure 2B.
HEK293T cells were transfected with wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) Casp11-mCherry and imaged by fluorescence microscopy 18 hr post-transfection. Nuclei (blue) were stained with Hoechst.
Source data for Figure 2D.
HEK293T cells were transfected with Casp11-mCherry constructs and 6 hr post-transfection, cells were incubated with increasing amounts of pan-caspase inhibitor zVAD (0–200 μM; twofold increments). Whole-cell lysates were isolated 12 hr post-transfection and immunoblotted for mCherry or β-actin as loading control as indicated. Cleaved p10-mCherry is denoted.
Source data for Figure 2E.
Casp11-mCherry speck formation was assayed in zVAD-treated cells by fluorescence microscopy. Nuclei are stained with Hoechst.
Source data for Figure 2F.
Speck formation in Figure 2E was quantified as percentage of Casp11-mCherry-expressing cells containing at least one speck. Dose–response curves were plotted by least-squares nonlinear regression ([Log2(inhibitor) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(X-LogIC50)); R2 indicated).
Gasdermin D (GSDMD) expression plasmid was co-transfected with increasing doses of indicated Casp11 constructs in HEK293T cells. WT untagged Casp11 was included as positive control. After 12 hr (A) …
Source data for Figure 2—figure supplement 1A.
Gasdermin D (GSDMD) expression plasmid was co-transfected with increasing doses of indicated Casp11 construct in HEK293T cells. WT untagged Casp11 was included as positive control.
Wild-type (B6) or Casp11-/- primary BMDMs were primed with Pam3CSK4 for 4 hr, followed by transfection with the indicated concentrations of LPS from S. enterica serotype Minnesota. To inhibit Casp11 …
Source data for Figure 2—figure supplement 2C.
Immunoblot for gasdermin D (GSDMD) cleavage in supernatants (sup) and whole-cell lysates (XT). β-actin is indicated as loading control.
(A) Tagged caspase-11 expression used for FLAG-based co-immunoprecipitation. (B) HEK293T cells were transiently transfected with 2X-FLAG-tagged wild-type (WT) or catalytically inactive (C254A) …
Source data for Figure 2—figure supplement 3B.
HEK293T cells were transiently transfected with 2X-FLAG-tagged wild-type (WT) or catalytically inactive Casp11 expression plasmids alongside WT or C254A mCherry-tagged Casp11 (5 μg). 48 hr post-transfection, whole-cell lysates were immunoprecipitated by anti-FLAG antibodies as described in ‘Materials and methods,’ and immunoblotted for mCherry, FLAG, or GAPDH as a loading control, as indicated.
(A) HEK293T cells stably expressing 2xFLAG-Casp11 (WT) or 2xFLAG-Casp11 (C254A) were transfected with LPS (1 μg/mL) from S. enterica serovar Minnesota and imaged by fluorescence microscopy 24 hr …
Source data for Figure 3B.
2xFLAG-Casp11 protein levels in each stable HEK293T cell line were determined by immunoblotting for FLAG. β-actin was used as loading control.
(A) Schematic representing experimental design of colocalization experiment (created with Biorender.com). (B) HEK293T cells stably expressing wild-type (WT) or catalytically inactive (C254A) …
Source data for Figure 3—figure supplement 1B.
HEK293T cells stably expressing wild-type (WT) or catalytically inactive (C254A) 2xFLAG-Casp11 were transiently transfected with mCherry-tagged WT or C254A Casp11 constructs for 24 hr. Stably expressed 2xFLAG-Casp11 was stained by immunofluorescence using anti-FLAG (yellow) and cells were imaged by confocal microscopy (×63 objective). Nuclei (blue) are stained with DAPI.
(A, E) Schematic diagram indicating co-transfection combinations used in (B–H). Untagged full-length wild-type (WT) caspase-11 gene constructs were transfected at increasing doses, together with a …
Source data for Figure 4B.
12 hr post-transfection of indicated plasmids, whole-cell lysates were harvested and immunoblotted for mCherry or β-actin as a loading control.
Source data for Figure 4C.
Untagged full-length wild-type (WT) caspase-11 gene constructs were transfected at increasing doses, together with a fixed amount of indicated mCherry-tagged Casp11. 18 hr following transfection, the cells were imaged by fluorescence microscopy. Nuclei (blue) were stained with Hoechst.
Source data for Figure 4D.
Speck formation in Figure 4C was quantified as percentage of Casp11-mCherry-expressing cells containing at least one speck. Dose–response curves were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(LogEC50-X))).
Source data for Figure 4F.
Whole-cell lysates of indicated transfected cells were harvested and immunoblotted for mCherry. β-actin was used as loading control.
Source data for Figure 4G.
HEK293T cells transfected with indicated Casp11 constructs were imaged by fluorescence microscopy. Nuclei (blue) were stained with Hoechst.
Source data for Figure 4H.
Speck formation in Figure 4G was quantified as percentage of Casp11-mCherry-expressing cells containing at least one speck. Dose–response curves were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y=Bottom + (Top-Bottom)/(1+10(LogEC50-X))).
(A) Schematic indicating plasmids used in co-transfection studies. (B, C) Unlabeled wild-type (WT) Casp11 was transfected into HEK293T cells at increasing doses, together with a fixed dose (0.25 μg) …
Source data for Figure 4—figure supplement 1B.
Unlabeled wild-type (WT) Casp11 was transfected into HEK293T cells at increasing doses, together with a fixed dose (0.25 μg) of indicated mCherry-tagged Casp11 construct and a fixed dose of murine gasdermin D (GSDMD, 0.05 μg). 14 hr post-transfection, Casp11-mediated cytotoxicity was measured by determining percent lactate dehydrogenase (LDH) release. Dose–response curves in (B) were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(LogEC50-X))).
Source data for Figure 4—figure supplement 1C.
HEK293T whole-cell lysates and supernatants (sup) from Figure 4—figure supplement 1B were immunoblotted for gasdermin D (GSDMD). β-actin is indicated as loading control.
(A) Schematic diagram indicating co-transfection combinations used in (B–D). Unlabeled wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) caspase-11 constructs were transfected …
Source data for Figure 5B.
Unlabeled wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) caspase-11 constructs were transfected into HEK293T cells at increasing doses, together with a fixed dose of catalytically inactive (C254A) mCherry-tagged caspase-11. 12 hr post-transfection, whole-cell lysates were harvested and immunoblotted for mCherry or β-actin as loading control.
Source data for Figure 5C.
Unlabeled wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) caspase-11 constructs were transfected into HEK293T cells at increasing doses, together with a fixed dose of catalytically inactive (C254A) mCherry-tagged caspase-11. 18 hr following transfection, cells were imaged by fluorescence microscopy. Nuclei (blue) are stained with Hoechst.
Source data for Figure 5D.
Speck formation in (C) was quantified as percentage of mCherry-expressing cells containing at least one speck. Dose–response curves were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(LogEC50-X))).
Source data for Figure 5E.
HEK293T cells stably expressing human gasdermin D were transiently transfected with a fixed dose of empty plasmid (Veh) OR mCherry-tagged C254A caspase-11, plus increasing doses of unlabeled wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) caspase-11 constructs. Cytotoxicity was measured as percent lactate dehydrogenase (LDH) release 18 hr post-plasmid transfection (with respect to 1% Triton X-100-induced cytotoxicity). Dose–response curves were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(LogEC50-X)); R2 indicated).
(A) Schematic representation of fluorescent Casp11 constructs that allow for inducible dimerization by the chemical dimerizer AP20187 (created with BioRender.com). (B) HEK293T cells were transfected …
Source data for Figure 6B.
HEK293T cells were transfected with WT or catalytically inactive (C254A) DmrB-(ΔCARD)-Casp11-FLAG-mCherry constructs. 24 hr post-transfection, cells were incubated with AP20187 (1 μM) for 6 hr and imaged by confocal microscopy. Nuclei (blue) are stained with Hoechst.
Source data for Figure 6D.
HEK293T cells stably expressing human gasdermin D (HEK293ThGSDMD) were transfected with WT or catalytically inactive (C254A) DmrB-(ΔCARD)-Casp11-FLAG-mCherry constructs, and incubated in AP20187 (1 μM) for 6 hr. Lysates were harvested and immunoblotted for GSDMD and mCherry, with GAPDH as loading control.
HEK293T cells were transiently co-transfected with ΔCARD DmrB-Casp11-FLAG-mCherry constructs (0.1 μg; Figure 4A) and murine gasdermin D (GSDMD, 0.01 μg) for 24 hr before the addition of AP20187 (1 …
Source data for Figure 6—figure supplement 1.
HEK293T cells were transiently co-transfected with ΔCARD DmrB-Casp11-FLAG-mCherry constructs (0.1 μg; Figure 4A) and murine gasdermin D (GSDMD, 0.01 μg) for 24 hr before the addition of AP20187 (1 μM) for 6 hr. Casp11 protein expression and cytotoxicity were determined by immunoblotting for mCherry and GSDMD in pooled lysates and supernatants. GAPDH is indicated as loading control. FL = full-length; CL = cleaved.
(A) HEK293T cells were transfected with indicated Casp11-mCherry constructs. 18 hr post-transfection, cells were fixed and prepared for microscopy. Nuclei are stained with Hoechst. White arrows …
Source data for Figure 6—figure supplement 2A.
HEK293T cells were transfected with indicated Casp11-mCherry constructs. 18 hr post-transfection, cells were fixed and prepared for microscopy. Nuclei were stained with Hoechst.
Source data for Figure 6—figure supplement 2D.
The fluorescent reporter citrine was fused to the C-terminus of wild-type (WT) or CARD-only Casp11, transfected into HEK293T cells as indicated, and imaged by confocal microscopy 18 hr post-transfection. Nuclei (magenta) were stained with DRAQ7, cytosolic content (blue) was stained with cell tracer violet.
(A) Schematic indicating Casp11-mCherry constructs that allow for inducible processing by tobacco etch virus (TEV) protease. The TEV protease consensus cleavage sequence (ENLYFQ/G) replaced the …
Source data for Figure 7B.
HEK293T cells were transfected with the indicated TEV-cleavable Casp11-mCherry constructs, together with increasing doses of V5-TEV protease (0–500 ng/well). Whole-cell lysates were harvested 12 hr post-transfection and immunoblotted for mCherry or β-actin (loading control).
Source data for Figure 7D.
HEK293T cells from (B) were imaged by fluorescence microscopy and speck formation was quantified as percentage of Casp11-mCherry-expressing cells containing at least one speck. Dose–response curves were plotted by least-squares nonlinear regression ([Log2(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10(LogEC50-X)); R2 indicated).
Tables of plasmids, cell lines and oligonucleotides used in this study.