Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: (A and B) The indicated THP-1 cells were treated with VbP (10 μM), MeBs (10 μM), CHR-2797 (10 μM), or batimastat (10 μM) for 6 h before LDH release and immunoblot analyses. (C and D) OCI-AML2 or resting human CD3+ T cells were treated with VbP (10 μM) and/or MeBs (10 μM) for (C) 6 h or (D) 18 h before LDH release and immunoblot analyses. (E and F) THP-1 or MV4;11 cells were treated with VbP (10 μM), CQ31 (20 μM), and/or MeBs (10 μM) for 16 h before LDH release and immunoblot analyses.

(G) WT or Casp1−/− RAW 264.7 cells were treated with VbP (10 μM) and/or MeBs (10 μM) for 6 h before LDH release and immunoblot analyses. (H and I) WT or NLRP1−/− human N/TERT-1 keratinocytes were treated with VbP (0.2 μM), MeBs (20 μM), or CQ31 (20 μM) for 24 h before IL-1β release and immunoblot analyses. Data are means ± SEM of three biological replicates. All data, including immunoblots, are representative of three or more independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by two-sided Student’s t test. n.s., not significant. See also Figure S1.

Article Snippet: Batimastat (Batim.) , Tocris , 2691.

Techniques: Western Blot

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: (A–C) Inhibition of the indicated peptidase activity in CARD8−/− THP-1 (A, B) or HEK 293T (C) cells by VbP (10 μM), MeBs (10 μM), Batimastat (Batim., 10 μM), or CHR-2797 (10 μM). (D) HEK 293T cell lysates (0.5 mg/mL) were incubated with the peptide PASKYLF (1 mM) and VbP (10 μM) or MeBs (10 μM) for 6 h prior to quantitation of peptide cleavage by an alanine release assay. (E) HEK 293T cells were treated with VbP (10 μM), MeBs (10 mM), or bortezomib (10 μM) for 6 h. Intracellular metabolites were extracted and the indicated dipeptide concentrations were measured by LC-MS. (F) HEK 293T cells were pretreated with vehicle (DMSO) or Bort. (10 μM) for 30 min, then treated with vehicle (DMSO) or MeBs (10 μM) for 5.5 h. Intracellular metabolites were extracted and dipeptide concentrations were measured by LC-MS. Data are means ± SEM of three or more biological replicates. Peptidase activity data are representative of three or more independent experiments, while endogenous peptide measurement data were collected in a single experiment. ***p < 0.001 by two-sided Student’s t test. n.s., not significant. See also Figure S4.

Article Snippet: Batimastat (Batim.) , Tocris , 2691.

Techniques: Inhibition, Activity Assay, Incubation, Quantitation Assay, Release Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: (A) WT or SMAC−/−/HTRA2−/− THP-1 cells were treated with MeBs (10 μM), Batimastat (10 μM), or BV6 (5 μM) for 24h prior to immunoblot analysis. (B) Chemical structures of MeBs (top) and CQ83 (bottom). (C) THP-1 cells were treated with MeBs (1 μM), CQ83 (1 μM), and/or VbP (10 μM), and cell viability was assessed by CellTiter-Glo (CTG) after 6 h. Data are means ± SEM of four biological replicates. (D and E) Scatterplot (D) and immunoblots (E) depict the proteins enriched by CQ83 and competed by MeBs as determined by TMT-MS (D) or immunoblotting (E).

(F) CETSA analyses of bestatin (10 μM), Batimastat (10 μM), BV6 (5 μM), and GDC-0152 (5 μM) in HEK 293T cell lysates. (G) MV4;11 cells were treated with the indicated aminopeptidase inhibitors or IAP agonists (BV6, GDC-0152) for 24 h prior to immunoblotting analysis. All compounds were treated at 10 μM except the following: fumagillin (3 μM); compound 5385 (DPP7 inhibitor; 20 μM), compound 8j (selective DPP8/9 inhibitor; 20 μM), and sitagliptin (DPP4 inhibitor; 20 μM), VbP (2 μM), BV6 (5 μM), and GDC-0152 (5 μM). (H) MV4;11 cells were treated with VbP (10 μM), MeBs (10 μM), and/or GDC-0152 (5 μM) for 6 h prior to LDH and immunoblot analyses. (I) CARD8−/− THP-1 cells stably containing doxycycline (DOX)-inducible CARD8 E274R were induced with 100 ng/mL DOX for 16 h prior to treatment with DMSO, MeBs (10 μM) or GDC-0152 (5 μM) for 6 h. Samples were then collected for LDH release and immunoblot analyses. (J) The proposed biological impacts of MeBs. MeBs inhibits APs, resulting in blockade of amino acid recycling and peptide accumulation. Some peptides bind to and thereby degrade cIAP1, some inhibit the N-end rule pathway, and some trigger CARD8/NLRP1 NT degradation. The peptides that modulate each of these effects are likely (at least partially) distinct. All data, including immunoblots, are representative of three or more independent experiments. ***p < 0.001, *p < 0.05 by two-sided Student’s t test. n.s., not significant. See also Figure S5 and Table S4.

Article Snippet: Batimastat (Batim.) , Tocris , 2691.

Techniques: Western Blot, Stable Transfection

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Batimastat (Batim.) , Tocris , 2691.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Batimastat (Batim.) , Tocris , 2691.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software