Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Treatment of rat cortical microglia with TNF-α or LPS up-regulates, in a time-dependent manner mRNA for Saa1 but not Saa3 . ( a ) SAA1; ( b ) SAA3. Cultures of microglia were treated the day after plating (DMEM + 0.5% FCS) with 10 ng/ml TNF-α or 100 ng/ml LPS and processed 1, 3, 6 and 24 h later for qRT-PCR, as detailed in Methods. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) at each time point for the control (untreated) cultures and are means ± s.e.m. n = 6. * p < 0.05 vs control (CTRL); ** p < 0.01 vs control; *** p < 0.001 vs control. # p < 0.05 vs control and TNF-α; ## p < 0.01 vs control and TNF-α; ### p < 0.001 vs control.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Treatment of enriched rat cortical astrocytes with TNF-α or LPS up-regulates, in a time-dependent manner mRNA for Saa1 but not Saa3 . ( a ) SAA1; ( b ) SAA3. Cultures were treated the day after plating (DMEM + 0.5% FCS) with 10 ng/ml TNF-α or 100 ng/ml LPS and processed 1, 3, 6 and 24 h later for qRT-PCR, as detailed in Methods. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) at each time point for the control (untreated) cultures and are means ± s.e.m. n = 3. *** p < 0.001 vs control; ### p < 0.001 vs control and TNF-α.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Addition of microglia to L-leucyl-L-leucine methyl ester (L-LME)-treated astrocytes fails to fully restore TNF-α and LPS-induced increases in Saa1 mRNA. Enriched cortical astrocytes (250,000 per 24-well) were treated with 50 mM L-LME for 60 min, and returned to fresh culture medium for 24 h. After this time (medium change to DMEM + 0.5% FCS) purified cortical microglia were added, at the numbers indicated on the horizontal axis (+5 K, + 10 K, + 20 K, + 250 K) to the astrocyte cultures and incubation continued for a further 24 h. Cultures were then challenged with either 10 ng/ml TNF-α or 100 ng/ml LPS and cells processed for Saa1 mRNA expression by qRT-PCR 6 h ( a ) and 24 h ( b ) later. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) at each time point and are means ± s.e.m. n = 3. *p < 0.05 vs control (CTRL); **p < 0.01 vs control; ***p < 0.001 vs control. ## p < 0.01 vs TNF-α; ### p < 0.001 vs TNF-α. Similar results were obtained in a second experiment. ( c ) The same numbers of microglia were cultured in a parallel 24-well plate and subjected to the same treatments as above and then processed for Saa1 mRNA expression by qRT-PCR at 24 h. For comparison, samples plated with 250,000 microglia per well were also analyzed. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) and are means ± s.e.m. n = 3. ***p < 0.001 vs all other cell densities; ### p < 0.001 vs TNF-α.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Purification, Incubation, Expressing, Quantitative RT-PCR, Cell Culture, Comparison

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Recombinant human Apo-SAA or LPS treatment of rat cortical enriched astrocytes for 24 h increases the production of IL-1β ( a ), TNF-α ( b ) and NO ( c ). Cultures were treated the day after plating (DMEM + 0.5% FCS) with 0.5 or 1.5 μg/ml recombinant human Apo-SAA (Apo-SAA) or 100 ng/ml LPS and culture medium collected 24 h later for measurement of TNF-α, IL-1β by ELISA and NO by Griess reaction, as detailed in Methods. Data are expressed as means ± s.e.m. n = 3. *** p < 0.001 vs control (CTRL). ## p < 0.01 vs 1.5 µg/ml Apo-SAA; ### p < 0.001 vs 1.5 µg/ml Apo-SAA.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Recombinant human Apo-SAA or LPS treatment of purified rat cortical microglia for 24 h increases the production of TNF-α ( a ), IL-1β ( b ), and NO ( c ). Cultures were treated the day after plating (DMEM + 0.5% FCS) with 1.5 μg/ml recombinant human Apo-SAA (Apo-SAA) or 100 ng/ml LPS and culture medium collected 24 h later for measurement of TNF-α, IL-1β by ELISA and NO by Griess reaction, as detailed in Methods. Data are expressed as means ± s.e.m. n = 3. *** p < 0.001 vs control (CTRL). ## p < 0.01 vs 1.5 µg/ml Apo-SAA.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Recombinant, Purification, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: CLI-095 abolishes Apo-SAA- and LPS-stimulated production of TNF-α ( a ) and NO ( b ) by rat cortical enriched astrocytes (▓ ) and purified microglia (■) treated with recombinant human Apo-SAA or LPS for 24 h. Cultures were treated the day after plating (DMEM + 0.5% FCS) with 0.5 or 1.5 μg/ml recombinant human Apo-SAA ('SAA') or 100 ng/ml LPS, together with 0.5 μg/ml CLI-095 (‘CLI’). Culture medium was collected 24 h later for measurement of TNF-α ( a ) and NO ( b ) ELISA, as detailed in Methods. Data are expressed as means + s.e.m. n = 3. ***p < 0.001 vs control (CTRL). Similar results were obtained in a second experiment.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

doi: 10.1038/s41598-017-12529-7

Figure Lengend Snippet: Treatment of rat cortical enriched astrocytes ( a ) and purified microglia ( b ) with recombinant human Apo-SAA or LPS for 24 h up-regulates expression of Saa1 mRNA. Cultures were treated the day after plating (DMEM + 0.5% FCS) with the indicated concentrations (μg/ml) of recombinant human Apo-SAA ('SAA') ( a , b ) or 100 ng/ml LPS ( a ), together with 0.5 μg/ml CLI-095 (‘CLI’) (where indicated) ( a ) and processed 24 h later for qRT-PCR, as detailed in Methods. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) and are means ± s.e.m. n = 3. * p < 0.05 vs control (CTRL); *** p < 0.001 vs CTRL. Similar results were obtained in a second experiment.

Article Snippet: Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, lipopolysaccharide (LPS; Ultra-Pure LPS-EB from E. coli 0111:B4 strain) and ethyl-(6 R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France), Griess reagent, papain, DNase I (bovine pancreas), trypsin inhibitor, L-LME, recombinant human fibroblast growth factor-2 (FGF2), recombinant rat TNF-α (T5944, cell culture tested, endotoxin level <0.1 ng/µg as determined by the Limulus amebocyte lysate method), recombinant human platelet-derived growth factor AA and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (London, UK); Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy).

Techniques: Purification, Recombinant, Expressing, Quantitative RT-PCR

Journal: Immunity

Article Title: Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock

doi: 10.1016/j.immuni.2018.06.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: At 24 h post-plating cells were primed with 5 μg/ml poly(I:C) (HMW, InvivoGen) and then transfected with LPS from E. coli 0111:B4 (2 μg/ml, LPS-EB Ultrapure, InvivoGen) using 0.25% v/v FuGENE HD (E2311, Promega).

Techniques: Labeling, Plasmid Preparation, Recombinant, In Vivo, Molecular Weight, Cell Culture, Cell Viability Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Schematic illustration of the dynamic organellar maps workflow to dissect inflammasome activation upstream of NLRP3 in immortalized bone-marrow derived macrophages (iBMDMs) deficient in Caspase-1 or ASC. After activation and isotonic lysis, SILAC heavy labelled (Lys8, Arg10) iBMDMs are subjected to incremental ultracentrifugation, enriching different organellar fractions prior to addition of a light-labelled (Lys0, Arg0) whole-organellar reference spike-in. (B) Principal component analysis (PCA) of sub-organellar fractionation proteomes after TLR4-stimulation (priming, LPS 1 µg/ml, 4 h) or NLRP3 (LPS 4 h + Nigericin 2 µM/ R837 60 µg/ml for 120 min) stimulation in Caspase-1 -/- Balb/C iBMDMs. (C) Profile plot showing relative ASC distribution over sub-organellar fractions in Caspase-1 -/- iBMDMs. (D) Enrichment analysis for gene ontology cellular compartment (GOCC) terms and Uniprot keywords using Fisher exact test on proteins significantly changing (p < 0.05; FDR < 0.02) localization upon NLRP3 activation in ASC -/- or Caspase-1 -/- iBMDMs. (E) Profile plots showing relative distribution of trans-Golgi-network proteins Tgoln1; Tgoln2, B4galnt1, Glg1 and Nucb1, as well as all clathrin or AP2 complex related proteins across organellar fractions from TLR4- or TLR4 and NLRP3-activated ASC -/- iBMDM. (F) 3D-Principal-component-analysis of median normalized intensities per fraction showing position of median AP2-complex subunits (grey) in LPS-primed control with annotations for endosomal (red), Golgi- (orange) or plasma membrane proteins (blue). (G) 3D-Principal-component-analysis of median normalized intensities per fraction showing relocalization of AP2-complex subunits (grey-LPS, black-Nigericin, movement indicated by lines) upon 2 µM Nigericin treatment with annotations for endosomal (red), Golgi- (orange) or plasma membrane proteins (violet). (H) As in (H) for 60 µg/ml R837. (I) IL-1β release of wt C57Bl6 BMDMs in Hank’s balanced salt solution (HBSS) (5 mM KCl) or K + -free HBSS (0 mM KCl, + 5 mM NaCl) ± 2 µM monensin. Statistics indicate significance by student’s two-tailed t-test (*** = P < 0.001). (J) Enrichment analysis of GOCC terms and Uniprot keywords using a Fisher exact test on all proteins significantly (p < 0.05; FDR < 0.02) changing localization upon monensin treatment or exposure to K + -free HBSS in ASC -/- or Caspase-1 -/- iBMDMs. (K) Heatmap showing combined absolute fold-changes of organellar marker proteins belonging to mitochondria, TGN, AP2 or lysosomes from dynamic organellar maps in ASC-deficient BMDMs treated with LPS (control), 2 µM Nigericin, 2 µM Nigericin + 80 µM KCl, 2 µM monensin or 2 µM monensin in K + -free buffer.

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Activation Assay, Derivative Assay, Lysis, Multiplex sample analysis, Fractionation, Control, Clinical Proteomics, Membrane, Two Tailed Test, Marker

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Number of protein groups identified in Caspase-1 -/- iBMDMs before and after filtering for variance and 100% data completeness. (B) Principal component analysis of median normalized protein group L/H intensities with annotations for organellar reference proteins, exported from our webapp for LPS-treated and LPS+Nigericin treated Caspase-1-deficient macrophages. Colors indicate organellar annotations according to . (C) Heatmap of mean absolute fold change per fraction of proteins that change significantly compared to LPS control in 3 out of 5 replicates entailing both Nigericin and R837 maps. (D) Enrichment analysis for gene ontology cellular compartment (GOCC), - biological process (GOBP), -molecular function or KEGG terms and Uniprot keywords using Fisher exact test on proteins significantly (p < 0.05; FDR < 0.02, absolute fold change compared to control > 0.2) changing localization upon K + -depletion in ASC -/- iBMDMs. (E) as in (D) for 10 µM monensin stimulation.

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Control