Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat

doi: 10.1161/HYPERTENSIONAHA.116.08826

Figure Lengend Snippet: Urinary Umod peptides are increased in the stroke-prone spontaneously hypertensive rat (SHRSP) relative to Wistar–Kyoto (WKY) in a pregnancy-dependent manner. Seven peptides detected in the urinary peptidome were derived from Umod protein ( A ). Of these peptides, they were all increased in a pregnancy-specific manner in SHRSP (n=7) relative to WKY (n=7) at gestational days (GDs) 12, GD18, or GD12 and GD18. Taking into account the sum of all of the 7 peptides ( B ) showed that Umod peptides were increased in a pregnancy-specific manner at GD12 and GD18 in SHRSP relative to WKY (* P <0.05, ** P <0.01 vs WKY analyzed by Wilcoxon rank test).

Article Snippet: Gene expression assay for uromodulin ( Umod ) in kidney tissues was performed using the following probes from Thermo Fisher, Paisley, United Kingdom: Umod (Rn01507237_m1) and Actb (4352340E).

Techniques: Derivative Assay

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat

doi: 10.1161/HYPERTENSIONAHA.116.08826

Figure Lengend Snippet: Increase in Umod in stroke-prone spontaneously hypertensive rat (SHRSP) validated in urine and kidney tissue. A , Purified Umod from the urine of Wistar–Kyoto (WKY; n=7) and SHRSP (n=7) was run on 10% NuPAGE gel and blotted onto a single polyvinylidene fluoride membrane. Umod showed an increase in SHRSP in pregnancy-dependent manner at gestational days (GDs) 12 and 18. B , Gene expression of Umod was measured in kidney tissue from nonpregnant and pregnant (GD18) WKY and SHRSP (n=5). Umod expression was increased in kidney tissue from SHRSP at both NP ( P =n.s.) and GD18 time points ( P <0.001). C , Umod protein was measured from kidney tissue extract of pregnant (GD18) SHRSP (n=4) and WKY (n=4). Pregnant SHRSP showed increased Umod expression in kidney tissue (** P <0.01, *** P <0.001 vs WKY analyzed by 1-way ANOVA, 2-way ANOVA, and t test).

Article Snippet: Gene expression assay for uromodulin ( Umod ) in kidney tissues was performed using the following probes from Thermo Fisher, Paisley, United Kingdom: Umod (Rn01507237_m1) and Actb (4352340E).

Techniques: Purification, Membrane, Gene Expression, Expressing

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat

doi: 10.1161/HYPERTENSIONAHA.116.08826

Figure Lengend Snippet: A , Schematic representation of rat Umod structure containing an epidermal growth factor–like domain (orange box I, II, and III), the Zona Pellucida (ZP) domain, internal and external hydrophobic patches (IHP and EHP, respectively), hepsin cleavage site and glycosylphosphatidylinositol (GPI) anchoring site. The zoomed-in sequence represents the C-terminal region identified in mass spectrometry rat data (pink) and previous human studies on preeclampsia (blue). B , Deglycosylation of Umod identified 2 bands in untreated WKY (pool of n=7) and stroke-prone spontaneously hypertensive rat (SHRSP; pool of n=7) at all gestational day (GD), and only single band in nifedipine-treated SHRSP (pool of 3) at GD12 and GD18. C , In the polymerization assay, the pellet fraction (P) represents the polymerization-competent and supernatant (S) the polymerization-incompetent Umod. Polymerization assay showed polymerization-incompetent Umod in the supernatant (S) of untreated WKY (pool of n=7) and SHRSP (pool of n=7) at all GDs, whereas no Umod bands were observed in nifedipine-treated SHRSP (pool of 3). Polymerization-competent Umod in the pellet (P) was observed in untreated WKY and SHRSP, as well as in nifedipine-treated SHRSP. D , Proteasix software predicted few serine proteases and metalloproteases that might cleave the C-terminal of Umod, which resulted in the peptides observed in urine. CTSG indicates cathepsin G; ELANE, neutrophil elastase; GZMA, granzyme A; MEP, Meprin A subunit α; MMP, matrix metalloprotease; NP, nonpregnant; and PLG, plasminogen.

Article Snippet: Gene expression assay for uromodulin ( Umod ) in kidney tissues was performed using the following probes from Thermo Fisher, Paisley, United Kingdom: Umod (Rn01507237_m1) and Actb (4352340E).

Techniques: Sequencing, Mass Spectrometry, Polymerization Assay, Software

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