pma differentiated thp 1 cells  (InvivoGen)

Journal: bioRxiv

Article Title: NLRP11 promotes non-canonical inflammasome activation in human macrophages by enhancing caspase-4 recognition of cytosolic lipopolysaccharide

doi: 10.64898/2026.02.02.703338

Figure Lengend Snippet: ( A-B ). Cell death of THP-1 macrophages upon infection with S. flexneri 2457T ( A ), or electroporation with S. enterica serovar Minnesota LPS ( B ), measured by LDH release, as percentage of Triton X-100-induced cell lysis. N ≥ 4 biological replicates. ( C-D ). NLRP11 is required for the processing of CASP4 and gasdermin D (GSDMD) induced by S. flexneri infection ( C ), or S. enterica LPS electroporation ( D ). CASP4 is required for GSDMD processing induced by S. flexneri infection ( C ), or S. enterica LPS electroporation ( D ). Processing of CASP4 yields a p32 and/or p20 polypeptide, and processing of GSDMD yields a p30 polypeptide. Representative western blots. Molecular weight markers in kDa. DKO, NLRP11 −/− CASP4 −/− double knockout. Mean ± SEM. Two-way ANOVA with Tukey’s post hoc test *, P<0.05; **, P<0.01; ****, P<0.0001.

Article Snippet: PMA-differentiated THP-1 cells were primed with 1 μg/mL E. coli O111:B4 LPS (InvivoGen, tlrl-eblps) for 2.5 h and treated with 10 μM nigericin (InvivoGen, tlrl-nig) for 15 min prior to the assay when indicated.

Techniques: Infection, Electroporation, Lysis, Western Blot, Molecular Weight, Double Knockout

Journal: bioRxiv

Article Title: NLRP11 promotes non-canonical inflammasome activation in human macrophages by enhancing caspase-4 recognition of cytosolic lipopolysaccharide

doi: 10.64898/2026.02.02.703338

Figure Lengend Snippet: ( A-B ). Cell death of THP-1 macrophages upon infection with S. flexneri ( A ) or electroporation with E. coli O111:B4 LPS ( B ) measured by LDH release, as percentage of Triton X-100-induced cell lysis. Complementation of indicated THP-1 knockouts by transduction with indicated NLRP11 or CASP4 constructs. N ≥ 4 biological replicates. ( C ) Identification of CASP4 residues D174, and K19 KVR53-55 QEK62-64 as required for efficient processing of CASP4 and GSDMD induced by LPS electroporation. Representative western blots. Molecular weight markers in kDa. Mean ± SEM. Two-way ANOVA with Tukey’s post hoc test. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: PMA-differentiated THP-1 cells were primed with 1 μg/mL E. coli O111:B4 LPS (InvivoGen, tlrl-eblps) for 2.5 h and treated with 10 μM nigericin (InvivoGen, tlrl-nig) for 15 min prior to the assay when indicated.

Techniques: Infection, Electroporation, Lysis, Transduction, Construct, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: NLRP11 promotes non-canonical inflammasome activation in human macrophages by enhancing caspase-4 recognition of cytosolic lipopolysaccharide

doi: 10.64898/2026.02.02.703338

Figure Lengend Snippet: ( A-C ). LPS binding to anti-FLAG-coated plates pre-incubated with lysates of HEK293T cells expressing FLAG-NLRP11 and/or CASP4-FLAG. Addition of purified E. coli O111:B4 LPS ( A ), intact E. coli O126:H7 bacteria ( B ), or intact S. flexneri WT, Δ rfe , or Δ galU bacteria ( C ). Bound LPS was normalized to levels in the CASP4*-alone condition ( A ), CASP4* with E. coli O126:H7 ( B ), or CASP4* with WT S. flexneri ( C ). Panel C also shows the structure of LPS; vertical lines indicate the location of truncations in LPS produced by S. flexneri Δ rfe and S. flexneri Δ galU . ( D-E ) Increasing interaction between NLRP11 and CASP4 as a function of concentration of added HEK293T FLAG-NLRP11 lysate, without ( D ) or with ( E ) addition of LPS. FLAG-NLRP11 in transfected HEK293T cells bound to plates coated with purified WT CASP4 or indicated CASP4 derivatives at 10 μg/mL; detection of bound FLAG. N = 3 biological replicates. a.u., arbitrary units. ( F ) LPS binding to plates pre-incubated with lysates of HEK293T cells expressing FLAG-NLRP11 and indicated purified derivatives of CASP4*. ( G-H ) Levels of soluble cytosolic LPS (cLPS) not bound to macromolecular complexes. S. flexneri -infected THP-1 macrophages lysed with 0.02% digitonin. Shown is LPS within the separated and filtered cytosolic fractions. Quantified by LAL assay. CASP4*, catalytically inactive caspase. CASP4 K19E KVR QEK, LPS-binding defective caspase. CASP4 KVR, KVR53-55EEA. DKO, NLRP11 −/− CASP4 −/− double knockout. Mean ± SEM. Two-way ANOVA with Tukey’s post hoc test; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: PMA-differentiated THP-1 cells were primed with 1 μg/mL E. coli O111:B4 LPS (InvivoGen, tlrl-eblps) for 2.5 h and treated with 10 μM nigericin (InvivoGen, tlrl-nig) for 15 min prior to the assay when indicated.

Techniques: Binding Assay, Incubation, Expressing, Purification, Bacteria, Produced, Concentration Assay, Transfection, Infection, LAL Assay, Double Knockout