Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A THP-1 cells were incubated with or without (Ctrl) CLO at different doses for 1 h and then observed with a microscope. B The time-lapse images of THP-1 cells treated with CLO (100 nM). C The hemolytic activity of CLO at different doses was determined. D THP-1 cells were incubated with or without (Ctrl) CLO at different doses for 1 h, and then measured for LDH release. In panels A and B , arrows indicate membrane blebbing, and the scale bar is 30 μm. In panels C and D , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01(one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Microscopy, Activity Assay, Membrane

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A , B THP-1 cells were treated with CLO (100 nM) or necroptosis inducer TBZ (TNFα, the SMAC mimetic BV-6 and Z-VAD) in the presence of DMSO or the inhibitors GSK’963, GSK’872, and GW806742X (targeting RIPK1, RIPK3, and MLKL respectively) for 1 h or 16 h. The cells were then subjected to LDH release determination ( A ) and microscopy after PI staining ( B ). Scale bar, 30 μm. C THP-1 cells were treated with or without (Ctrl) CLO (100 nM) in the presence of Q-VD-OPh, Ac-YVAD-CMK, Ac-DEVD-CMK, Z-LEVD-FMK, Z-IETD-FMK, or DMSO for 1 h. LDH release was then measured. D THP-1 cells were treated with or without (Ctrl) CLO (100 nM) in the presence or absence of different concentrations of Ac-YVAD-CMK for 1 h. LDH release was then measured. E , F THP-1 WT and THP-1 GSDMD-KO cells were treated with or without (Ctrl) CLO (100 nM) or nigericin (Nig) for 1 h. LDH ( E ) and IL-1β ( F ) release was then determined. G THP-1 GSDMD-KO cells were treated as C and then measured for LDH release. ** p < 0.01. NS no significance (one-way ANOVA test A , C , D , and G or student’s unpaired t test E and F . Values are shown as means ± SD ( N = 3). N the number of replicates.

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Microscopy, Staining

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A J774A.1 cells were pretreated with MCC950, VX765, or DMSO for 1 h and then treated with CLO (CLO 100 nM) or ATP for 1 h. LDH release was then determined. B , C The cell lysate and supernatants from J774A.1 cells treated as above was immunoblotted with antibodies against Casp1, GSDMD, or β-actin (loading control). D , E PMA-differentiated THP-1 cells with or without (Null) deficiency in NLRP3 (NLRP3-KD) or Casp1 (Casp1-KD) were treated with or without (Ctrl) CLO (CLO 100 nM) or nigericin (Nig) for 1 h. LDH ( D ) and IL-1β ( E ) release was then determined. F The supernatant and the corresponding cell lysate from the above ( D , E ) treated J774A.1 cells were blotted with antibodies against Casp1, GSDMD, or β-actin (loading control). For panels A , D , and E , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01 (one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques:

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A , B J774A.1 cells pretreated with DCFH-DA were incubated with or without (Ctrl) DPI or NAC for 1 h. The cells were treated with or without CLO (10 nM) for 30 min. ROS production ( A ) and fluorescence intensity (λex, 488 nm; λem, 525 nm) ( B ) were then determined. C – G J774A.1 cells were pretreated with or without (−) DPI or NAC for 1 h and then treated with CLO (10 nM) or ATP for 1 h. Cell viability ( C ), LDH release ( D ), and IL-1β ( E ) release were determined. ASC speck (red) was detected by treating the cells with ASC-antibody and DAPI ( F ). The supernatant plus the corresponding cell lysate were blotted with antibody against Casp1, GSDMD, or β-actin (loading control) ( G ). H J774A.1 cells were incubated with or without (Ctrl) CLO (100 nM) or nigericin (Nig) for 30 min, and intracellular K + was then determined. I – K J774A.1 cells were pretreated with or without (−) different concentrations of KCl for 1 h, and then treated with or without (−) CLO (100 nM) for 1 h. LDH ( I ) and IL-1β release ( J ) was then determined. Immunoblot analysis of Casp-1, GSDMD, or β-actin was performed as above ( K ). Scale bars of panels A and F are 30 μm and 10 μm, respectively. For panels B – E and H – J , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01, * p < 0.05. NS no significance (one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Fluorescence, Western Blot

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A CLO was incubated with a membrane lipid strip spotted with 15 lipids, and the bound CLO was detected by immunoblotting. B THP-1 cells were incubated with or without (Ctrl) CLO (100 nM) or cholesterol (Cho.)-pretreated CLO for 1 h. CLO was localized by immunofluorescence microscopy using dyLight 650 anti-6×His tag antibody. Scale bar, 30 μm. C – E J774A.1 cells were treated with or without (Ctrl) CLO (100 nM), nigericin (Nig), or Cho-pretreated CLO or Nig for 1 h. LDH ( C ) and IL-1β ( D ) release was then determined, and Casp1 and GSDMD cleavage was determined by Western blot with antibodies against Casp1, GSDMD, and β-actin (loading control) ( E ). For panels C and D , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01. NS no significance (student’s unpaired t test).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Membrane, Stripping Membranes, Western Blot, Immunofluorescence, Microscopy

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A J774A.1 cells were treated with CLO (100 nM) or its mutants (100 nM) for 1 h, and LDH release was then determined. B Sterile defidrinated sheep blood was incubated with CLO (100 nM) or its mutants (100 nM) for 30 min and then detected for hemolysis. C A membrane lipid strip was incubated with the W477S-W479S mutant, and the bound protein was detected by immunoblotting. D THP-1 cells were incubated with or without (Ctrl) CLO (100 nM) or the W477S-W479S mutant (100 nM) for 1 h. The cells were stained with DAPI and subjected to immunofluorescence microscopy with dyLight 650 anti-6×His tag antibody. Scale bar, 30 μm. E , F J774A.1 cells were treated with or without (Ctrl) ATP, CLO (100 nM), or the W477S-W479S mutant (100 nM) for 1 h. The cells were determined for IL-1β release ( E ) and Casp1/GSDMD cleavage by immunoblot using antibodies against Casp1, GSDMD, and β-actin (loading control) ( F ). J774A.1 cells were treated with mutants (100 nM) for 1 h. LDH ( G ), IL-1β ( H ) and immunoblot analysis of Casp-1 and GSDMD ( I ) were assessed as above. ** p < 0.01. NS no significance (one-way ANOVA test A , B , G , and H or student’s unpaired t test E . Values are shown as means ± SD ( N = 3). N the number of replicates.

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Sterility, Incubation, Membrane, Stripping Membranes, Mutagenesis, Western Blot, Staining, Immunofluorescence, Microscopy