Journal: Redox Biology

Article Title: NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa -induced acute lung injury ☆

doi: 10.1016/j.redox.2022.102467

Figure Lengend Snippet: Effects of cigarette smoke on P. aeruginosa -triggered NLRP3 inflammasome activation. C57BL/6 adult male mice were exposed to RA, CS for 6 h or 2 weeks (a), or CS for 2 weeks (b–h). One hour after the last CS exposure, mice were given 1.0 × 10 5 CFU (determined based on the optical density of bacterial suspension) of PA103 (PA) in 50 μl saline or Ctrl (an equal volume of saline) by intratracheal instillation. The actual alive bacteria mice received were 1.8 × 10 5 CFU. 18 h after administration of PA, BAL fluid was collected for assessment of bacterial amount by counting colony forming units (CFU) (a). The data is presented as mean ± SD of Log10 (CFU/ml). 3–7 mice per experimental group were used. Lung homogenates from the experiment was prepared for assessment of NLRP3 inflammasome platform proteins (NLRP3, ASC, pro-caspase-1, and pro-IL-1β) and inflammasome activation (p20 caspase-1 and mature (cleaved) IL-1β) by immunoblot, using β-actin as a protein loading control (b). The relative levels of each protein of interest in each sample were determined by densitometry of their immunoblots normalized by immunoblots of respective protein loading controls. The densitometry values are presented as mean ± SD (c). 6–8 mice per experimental group were used. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. *p < 0.05. IL-1β levels in lung homogenates from this experiment were also assessed by ELISA (d). Data are presented as mean ± SD. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. **p < 0.01; ****p < 0.0001. The cytospun slides of BAL leukocytes (e) and cryosections of lung tissue (f, h) from these experiments were prepared for immunofluorescence microscopy to examine NLRP3 expression/localization (green) in either AM (CD68 + , red; e, f) or lung endothelial cells (von Willebrand factor (vWF+), red; h). DAPI was used to stain nuclei (blue). Panels e, f, h are representative images for each experimental group. Scale bar = 15 μm. The arrows in panels e–f indicate alveolar macrophages (M) and the arrows in panel h indicate lung endothelial cells (EC). The percentage of AM expressing NLRP3 over the total AM counted are presented as mean ± SD (g). Five slides at 40X and five slides at 100X per mouse lung tissue or BAL cytospun slides were used to count AM. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Caspase-1 inhibitor (Ac-YVAD-cmk) was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Expressing, Staining

Journal: Redox Biology

Article Title: NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa -induced acute lung injury ☆

doi: 10.1016/j.redox.2022.102467

Figure Lengend Snippet: Effects of pharmacological inhibition of caspase-1 on PA- and CS/PA-induced acute lung injury. C57BL/6 adult male mice were exposed to RA or CS for 2 weeks. One hour after the last CS exposure, mice were administered a caspase-1 inhibitor, Ac-YVAD-cmk (Ac–Y) at a dose of 10 mg/kg or vehicle intraperitoneally. One hour later, mice were given 1.0 × 10 5 CFU (determined based on the optical density of bacterial suspension) of PA103 (PA) in 50 μl saline or Ctrl (an equal volume of saline) by intratracheal instillation. The actual alive bacteria mice received were 3.5 × 10 5 CFU. 18 h after administration of PA, BAL fluid was collected for assessment of inflammasome activation by measuring mature IL-1β levels in cell-free BAL fluid by ELISA (a), BAL total protein levels (b), BAL inflammatory cells (c–f), BAL chemokine/cytokines (g–j), and BAL bacterial loads (k). Animal survival was recorded up to 36 h after administration of PA in a separate set of mice (l). 4–7 mice per experimental group were used. Data are presented as mean ± SD. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means for studies other than survival study, in which Log-rank (Mantel-Cox) test was used for determining difference among groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS: not significant.

Article Snippet: Caspase-1 inhibitor (Ac-YVAD-cmk) was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: Only enzymatically active FKBP38 interacts with Bcl-2. (A) Co-immunoprecipitation of endogenous FKBP38 and Bcl-2. After preincubation with 500 μM EGTA, SH-SY5Y cell lysate was incubated with rabbit anti-FKBP38 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using mouse anti-Bcl-2 antibody. Cell lysate preincubated with rabbit immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM calcium. This interaction was disrupted by 1 μM GPI1046. No interaction was observed in the absence of Ca2+. (B) SH-SY5Y crude cell extract was applied in the absence and presence of 20 μM CaM and 2 mM Ca2+ to maltose-binding protein-Bcl-2 fusion protein (MBP-Bcl-2) immobilized on amylose resin. After three washing steps, protein was eluted by 200 mM maltose and analyzed by Western blot using polyclonal anti-FKBP38 antibody. The proportion of active endogenous FKBP38 was quantified by Biorad Multi-Analyst software. (C) MBP-Bcl-2 was immobilized on amylose resin and incubated with (1) FKBP38, (2) FKBP38 and CaM, (3) FKBP38 and Ca2+/CaM and (4) FKBP38, Ca2+/CaM and 200 nM GPI1046. After washing, protein was eluted and analyzed by Western blot using polyclonal anti-FKBP38 antibody. (D) Inhibition of PPIase activity of 1 μM FKBP38 by Bcl-2 was measured in the PPIase assay in the presence of 5 mM CaCl2 and 5 μM CaM. The calculated Ki value was 0.74 μM.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Binding Assay, Software, Inhibition, Activity Assay

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 activity influences Bcl-2 function. (A) Near-UV CD spectra of 1 μM Ca2+/CaM/FKBP38 and 3 μM Bcl-2 were measured either separated (dotted line) or mixed (solid line) in a tandem cuvette. (B) Co-immunoprecipitation. SH-SY5Y cells were stimulated to apoptosis by 50 μM etoposide. Cell lysate preincubated with 500 μM EGTA was incubated with monoclonal mouse anti-Bcl-2 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using rabbit anti-FKBP38 antibody. Cell lysate preincubated with control mouse immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM Ca2+, whereas 1 μM GPI1046 abolished the interaction. Bcl-2/Bad interaction was investigated by incubation of SH-SY5Y cell lysate with hamster anti-Bcl-2 antibody and protein was detected using mouse anti-Bad antibody. Bad/Bcl-2 interaction was observed in the presence of 1 mM calcium and 1 μM GPI1046. In addition, SH-SY5Y cell lysate was incubated with monoclonal mouse anti-Bad antibody, and antibody/protein complexes formed were bound to protein G-Sepharose. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using hamster anti-Bcl-2 antibody. Cell lysate preincubated with mouse immunoglobin was used as a control. Bcl-2 bound to Bad in the presence of 1 mM Ca2+ and 1 μM GPI1046, indicating that GPI1046 interfered with the Bcl-2/FKBP38 interaction, allowing Bad/Bcl-2 complexes to form. (C) MBP-Bcl-2 fusion protein was immobilized on amylose beads and incubated with Bad (Sigma), FKBP38 and CaM either in the presence or absence of 2 mM Ca2+. Pellet (P) and supernatant (S) were subjected to 12.5% SDS–PAGE and analyzed by Western blotting using mouse anti-Bad antibody and hamster anti-Bcl-2 antibody. (D) Subcellular distribution of Bcl-2 and Bad in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with FITC-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody. Nuclei were stained with DAPI. Cells were treated for 16 h with 50 μM etoposide either in the absence or presence of 1 μM GPI1046. Cells transfected with FKBP38 RNAi construct were analyzed with ImageBlue-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and TRITC-conjugated goat anti-hamster antibody against hamster anti-Bcl-2. Nuclei were stained by 7-AAD. Because of better understanding, colors of this panel were adapted to other results presented in this figure. Again, inhibition of FKBP38 activity allows Bcl-2/Bad interaction after induction of apoptosis by 50 μM etoposide.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Activity Assay, Immunoprecipitation, Incubation, SDS Page, Western Blot, Immunostaining, Staining, Transfection, Construct, Inhibition

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 and Bcl-2 colocalize in neuroblastoma cells. (A) Localization of Bcl-2 and FKBP38 in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody and FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody. Cells were treated for 16 h with 50 μM etoposide and 2 μM GPI1046. Nuclei were stained with DAPI. (B) Subcellular distribution of FKBP38 in SH-SY5Y cells was studied by immunostaining with FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody and a subcellular structure localization kit (Chemicon).

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunostaining, Staining

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: (C) Subcellular localization of FKBP38 and Bcl-2 was studied by preparing mitochondria, ER, nucleus and cytosol from SH-SY5Y cells and analyzing these fractions by Western blotting using anti-FKBP38 and anti-Bcl-2 antibodies. Antibodies detecting cytochrome c (Cyt c) and FKBP13 were used as controls for mitochondrial and ER localization, respectively.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Western Blot