Journal: bioRxiv

Article Title: Leptospira interrogans prevents macrophage cell death and pyroptotic IL1β release through its atypical lipopolysaccharide

doi: 10.1101/2022.07.25.501344

Figure Lengend Snippet: A) Left panel . Western Blot analysis of caspase 3 in BMDMs after overnight infection with MOI 10-100 of L. interrogans serovar Manilae strain L495 or control stimulation during 5h with 1-5 μM of staurosporine. A) Right panel . Kinetics of caspase 3/7 activity, assessed by fluorometry measures every 15 min of cleaved substrate Ac-D EVD-AMC, in BMDMs after overnight infection with MOI 100 of L. interrogans serovar Manilae strain L495, or control stimulation during 5h with 1 μM of staurosporine. Dots correspond to mean +/− SD of technical replicates ( n =6). B) Western Blot and mRNA RT-qPCR analyses of caspase 11 in BMDMs after either 8h (WB, left panel ) or 6h (qPCR, right panel ) infection with MOI 100 of L. interrogans serovar Manilae strain L495 or control stimulation 1 μg/mL of E. coli LPS + 2-5 mM ATP. C) Western Blot analysis ( left panel ) and quantification of GSDMD ( right panel ) in BMDMs and RAW-ASC cells after 8h infection with MOI 100 of L. interrogans serovar Manilae strain L495 or control stimulation 1 μg/mL of E. coli LPS + 2-5 mM ATP. B-C) Bars correspond to the mean of at least three independent experiments. A-C) Data presented are representative of at least 3 independent experiments.

Article Snippet: Interaction between recombinant caspase 11 (Enzo) and E. coli LPS (Invivogen) or L. interrogans LPS from serovar Manilae strain L495 was addressed by binding assays performed by incubation of 100 ng of recombinant enzyme with 50-200 ng of LPS in PBS for 1 h at 37°C.

Techniques: Western Blot, Infection, Activity Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Leptospira interrogans prevents macrophage cell death and pyroptotic IL1β release through its atypical lipopolysaccharide

doi: 10.1101/2022.07.25.501344

Figure Lengend Snippet: A) Epifluorescence microscopy of RAW-ASC cells after 6h transfection with pCMV-GFP and either 10 μg/mL E. coli LPS or 10 μg/mL L. interrogans LPS from serovar Manilae strain L495. B) Cell death monitored by flow cytometry analysis of propidium iodide (PI) integration in RAW-ASC cells after 12h transfection with pCMV-GFP and either 10 μg/mL E. coli LPS or 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 . C) Cell death monitored by fluorimetry analysis of PI integration in RAW-ASC cells after 24h transfection with pcDNA3 and either 10 μg/mL E. coli LPS or 10 μg/mL L. interrogans LPS from serovar Manilae strain L495. Lines correspond to mean +/− SD of technical replicates ( n =4). D) LDH release measured by CyQuant assay on the supernatants of RAW-ASC cells after 24h transfection with pcDNA3 and either 10 μg/mL E. coli LPS or 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 (blue), serovar Icterohaemorrhagiae strain Verdun (red) or serovar Copenhageni strain Fiocruz L1-130 (green). Bars correspond to mean +/− SD of technical replicates ( n =6). E) Western Blot analysis in native conditions of caspase 11 in RAW-ASC cells after 24h transfection with pCMV-GFP and either 10 μg/mL E. coli LPS or 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 . A-E) Data presented are representative of at least 3 independent experiments.

Article Snippet: Interaction between recombinant caspase 11 (Enzo) and E. coli LPS (Invivogen) or L. interrogans LPS from serovar Manilae strain L495 was addressed by binding assays performed by incubation of 100 ng of recombinant enzyme with 50-200 ng of LPS in PBS for 1 h at 37°C.

Techniques: Epifluorescence Microscopy, Transfection, Flow Cytometry, CyQUANT Assay, Western Blot

Journal: bioRxiv

Article Title: Leptospira interrogans prevents macrophage cell death and pyroptotic IL1β release through its atypical lipopolysaccharide

doi: 10.1101/2022.07.25.501344

Figure Lengend Snippet: A) Left panels . Cell death monitored by flow cytometry and fluorimetry analysis of PI integration in RAW-ASC cells, 12h and 24h, respectively, after transfection with pCMV-GFP and 10 μg/mL E. coli LPS, 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 or 10 μg/mL of both LPS. A) Right panel . LDH release measured by CyQuant assay on the supernatant of RAW-ASC cells after 24h transfection with pcDNA3 and 10 μg/mL E. coli LPS + 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 (blue), serovar Icterohaemorrhagiae strain Verdun (red) or serovar Copenhageni strain Fiocruz L1-130 (green). Bars correspond to mean +/− SD of technical replicates ( n =6). B) Left panel . Western Blot analysis in native conditions of caspase 11 in RAW-ASC cells after 12h transfection with pCMV-GFP and 10 μg/mL E. coli LPS, 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 or 10 μg/mL of both LPS. B) Right panel . Western Blot analysis of GSDMD in RAW-ASC cells after 12h transfection with pCMV-GFP and 10 μg/mL E. coli LPS, 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 or 10 μg/mL of both LPS. C) Schematic representation of full length and recombinant caspase 11 lacking the LPS-binding CARD domain. D) Binding assay between recombinant caspase 11 and E. coli LPS or L. interrogans LPS from serovar Manilae strain L495, performed on highly reticulated 20% polyacrylamide native gels that do not allow the entrance of LPS and bound material and only allow migration of unbound proteins. E) LDH release measured by CyQuant assay on the supernatant of either RAW-ASC or parental RAW264.7 cells 24 h after transfection with pcDNA3 and 10 μg/mL E. coli LPS, 10 μg/mL L. interrogans LPS from serovar Manilae strain L495 or 10 μg/mL of both LPS. Bars correspond to mean +/− SD of technical replicates ( n =3). A-E) Data presented are representative of at least 3 independent experiments.

Article Snippet: Interaction between recombinant caspase 11 (Enzo) and E. coli LPS (Invivogen) or L. interrogans LPS from serovar Manilae strain L495 was addressed by binding assays performed by incubation of 100 ng of recombinant enzyme with 50-200 ng of LPS in PBS for 1 h at 37°C.

Techniques: Flow Cytometry, Transfection, CyQUANT Assay, Western Blot, Recombinant, Binding Assay, Migration

Journal: The Journal of Neuroscience

Article Title: A Molecular Platform in Neurons Regulates Inflammation after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.0157-08.2008

Figure Lengend Snippet: SCI induces activation and processing of caspase-1 and increases levels of ASC and caspase-11 but not NALP1. A–D, Representative immunoblot analysis of caspase-1 (A), caspase-11 (B), ASC (C), and NALP1 (D) in spinal cord lysates of sham (Sh) and traumatized rat cords at indicated times after injury. C shows that anti-ASC reacts specifically with ASC (26 kDa) and nonspecifically with proteins of ∼49 and 55 kDa (arrows, left). β-Tubulin was used as internal standard and control for protein loading. Data are presented as mean ± SEM. *p < 0.05, #p < 0.10 compared with sham. n = 5 per group.

Article Snippet: Proteins were resolved in 10–20% Tris-HCl Criterion precasted gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene difluoride membranes (Applied Biosystems, Foster City, CA), placed in blocking buffer [PBS, 0.1% Tween 20, and 0.4% I-Block (Applied Biosystems)], and then incubated for 1 h with the following: monoclonal antibody to caspase-11 (1:1000; Alexis Biochemicals), monoclonal antibody to caspase-1 (1:1000; Millipore), rabbit anti- Rattus novegicus , affinity-purified antibody ASC (1:10,000; Bethyl Laboratories), NALP1 (1:5000; Bethyl Laboratories), and NALP1 (1:1000; Abcam).

Techniques: Activation Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Molecular Platform in Neurons Regulates Inflammation after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.0157-08.2008

Figure Lengend Snippet: SCI induces association of NALP1 inflammasome proteins, processing of caspase-1, and cleavage of XIAP. Coimmunoprecipitation with ASC of sham lysates (Sh) and lysates obtained at 30 min, 6 h, and 3 d after SCI. ASC immunoprecipitates were blotted for ASC, caspase-1, caspase-11, NALP1, XIAP, and caspase-3 (control). In sham animals, anti-ASC immunoprecipitated (IP) NALP1, caspase-1, caspase-11, and the 53 kDa XIAP, thus indicating association of these proteins in a multiprotein complex. SCI induced increased association of NALP1 inflammasome proteins, processing of caspase-1 and caspase-11, and cleavage of XIAP into fragments. Preimmune serum did not immunoprecipitate inflammasome proteins and was used as control.

Article Snippet: Proteins were resolved in 10–20% Tris-HCl Criterion precasted gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene difluoride membranes (Applied Biosystems, Foster City, CA), placed in blocking buffer [PBS, 0.1% Tween 20, and 0.4% I-Block (Applied Biosystems)], and then incubated for 1 h with the following: monoclonal antibody to caspase-11 (1:1000; Alexis Biochemicals), monoclonal antibody to caspase-1 (1:1000; Millipore), rabbit anti- Rattus novegicus , affinity-purified antibody ASC (1:10,000; Bethyl Laboratories), NALP1 (1:5000; Bethyl Laboratories), and NALP1 (1:1000; Abcam).

Techniques: Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: A Molecular Platform in Neurons Regulates Inflammation after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.0157-08.2008

Figure Lengend Snippet: NALP1 inflammasome proteins are present in spinal cord motor neurons, and SCI induces alterations in protein expression patterns. Confocal images show motor neurons in the ventral horn of sham and injured spinal cords at 6 h after trauma. Sections were stained for caspase-1, caspase-11, ASC, and NALP1 (red) and the neuronal marker MAP2 (green). In sham animals, caspase-1 immunoreactivity was seen in the nucleus (arrow). By 6 h after injury, increased caspase-1 staining was present in neuronal nuclei, and patchy staining was present in the cell cytoplasm (arrow) and processes near the plasma membrane. Caspase-11 immunoreactivity showed diffuse punctate staining confined to the neuronal soma and processes (arrow). Increased caspase-11 staining was present by 6 h after trauma in the neuronal soma in a patchy distribution (arrow). Intense ASC and NALP1 staining was detected in the soma of spinal cord neurons and exhibited a patchy distribution pattern in the cytoplasm (arrow). Both inflammasome proteins showed increased expression as evidenced by intense patchy staining located near or associated with the plasma membrane (arrows) by 6 h after trauma. Scale bars, 20 μm.

Article Snippet: Proteins were resolved in 10–20% Tris-HCl Criterion precasted gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene difluoride membranes (Applied Biosystems, Foster City, CA), placed in blocking buffer [PBS, 0.1% Tween 20, and 0.4% I-Block (Applied Biosystems)], and then incubated for 1 h with the following: monoclonal antibody to caspase-11 (1:1000; Alexis Biochemicals), monoclonal antibody to caspase-1 (1:1000; Millipore), rabbit anti- Rattus novegicus , affinity-purified antibody ASC (1:10,000; Bethyl Laboratories), NALP1 (1:5000; Bethyl Laboratories), and NALP1 (1:1000; Abcam).

Techniques: Expressing, Staining, Marker

Journal: The Journal of Neuroscience

Article Title: A Molecular Platform in Neurons Regulates Inflammation after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.0157-08.2008

Figure Lengend Snippet: A, NALP1 inflammasome proteins are expressed in spinal cord neurons in culture. Spinal cord neurons (∼95% pure) were grown in culture for 14 d, harvested, and lysed. A segment (C5–C7) of adult rat spinal cord was excised and homogenized as outlined in Materials and Methods. Samples were immunoblotted for NALP1, ASC, caspase-1, caspase-11, and XIAP. Neurons in culture (culture); spinal cord tissue (cord). B, Activation of caspase-1 is induced in spinal cord neurons by treatment with the K+ ionophore valinomycin. Spinal cord neuronal cultures were grown for 14 d and treated for 4 h with 1 μm valinomycin or were left untreated (control). The cells were lysed and analyzed by immunoblot for caspase-1.

Article Snippet: Proteins were resolved in 10–20% Tris-HCl Criterion precasted gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene difluoride membranes (Applied Biosystems, Foster City, CA), placed in blocking buffer [PBS, 0.1% Tween 20, and 0.4% I-Block (Applied Biosystems)], and then incubated for 1 h with the following: monoclonal antibody to caspase-11 (1:1000; Alexis Biochemicals), monoclonal antibody to caspase-1 (1:1000; Millipore), rabbit anti- Rattus novegicus , affinity-purified antibody ASC (1:10,000; Bethyl Laboratories), NALP1 (1:5000; Bethyl Laboratories), and NALP1 (1:1000; Abcam).

Techniques: Activation Assay, Western Blot