Journal: Frontiers in Genetics

Article Title: Integrative analysis of single-cell and microarray data reveals SPI1-centered macrophage regulatory signatures in ulcerative colitis

doi: 10.3389/fgene.2025.1617834

Figure Lengend Snippet: SPI1(+) mainly regulates UC macrophage activation. (A) GSEA plots showing significant enrichment of macrophage activation-associated pathway in UC. (B) Transcription factors regulating macrophage activation-associated pathway including SPI1 (+). (C) Rank for regulons in macrophages based on the RSS. (D) Macrophages are highlighted in the UMA; (E) Binarized RAS for the top regulon SPI1 on UMAP. (F) UMAP showing SPI1 regulon activity across cell types. (G) Average expression of SPI1 regulon genes in macrophages. Data are expressed as mean ± SD. *P < 0.05, **P < 0 .01 compared to the other group.

Article Snippet: ELISA kit for IL-1β (ZC-37974, Zhuocai, China); ELISA kit for IL-10 (ZC-37962, Zhuocai, China); SPI1 antibody (55100-1-AP, Proteintech, China); iNOS antibody (22226-1-AP, Proteintech, China); Arg1 antibody (16001-1-AP, Proteintech, China); Tubulin antibody (80762-1-RR, Proteintech, China); Anti-rabbit IgG (H + L) (14780, Cell Signaling Technology, United States).

Techniques: Activation Assay, Activity Assay, Expressing

Journal: Frontiers in Genetics

Article Title: Integrative analysis of single-cell and microarray data reveals SPI1-centered macrophage regulatory signatures in ulcerative colitis

doi: 10.3389/fgene.2025.1617834

Figure Lengend Snippet: Identification of SPI1-regulated hub genes through the integration of gene set intersection and machine learning approaches. (A,B) LASSO regression analysis with cross-validation curve showing the optimal lambda value selection and coefficient profiles for gene selection. (C) Accuracy versus number of variables selected using RFE. (D) Bar chart of top genes ranked by importance scores. (E) SVM-RFE identified a 30-gene signature that achieved the highest classification accuracy. (F) Venn diagram illustrating the overlap between genes selected by three machine learning methods (LASSO, RFE-RF, and SVM-RFE). (G,H) Expression levels of SPI1, IRAK3, IL1RN, CD55, and PEA15 in two independent datasets GSE87466 (G) and GSE75214 (H) . (I) ROC curves for IRAK3, IL1RN, CD55 and PEA15 in the training dataset. (J) ROC curves for IRAK3, IL1RN, CD55 and PEA15 in the validation dataset. **** P < 0.0001 compared to the control group.

Article Snippet: ELISA kit for IL-1β (ZC-37974, Zhuocai, China); ELISA kit for IL-10 (ZC-37962, Zhuocai, China); SPI1 antibody (55100-1-AP, Proteintech, China); iNOS antibody (22226-1-AP, Proteintech, China); Arg1 antibody (16001-1-AP, Proteintech, China); Tubulin antibody (80762-1-RR, Proteintech, China); Anti-rabbit IgG (H + L) (14780, Cell Signaling Technology, United States).

Techniques: Biomarker Discovery, Selection, Expressing, Control

Journal: Frontiers in Genetics

Article Title: Integrative analysis of single-cell and microarray data reveals SPI1-centered macrophage regulatory signatures in ulcerative colitis

doi: 10.3389/fgene.2025.1617834

Figure Lengend Snippet: LPS-induced polarization of M1 macrophages is affected by SPI1 in vitro. (A–D) The protein expressions of SPI1 (B) , iNOS (C) , Arg1 (D) measured by WB. (E–G) Macrophage M1 polarization induced by LPS was detected by flow cytometry. (H, I) WB analysis was performed to assess the knockdown efficiency of SPI1 following si-SPI1 treatment, including three groups: untreated control, si-NC (non-targeting siRNA), and si-SPI1. (J–L) The expressions of iNOS (K) and Arg1 (L) measured by WB after SPI1 knockdown. (M–P) Macrophage polarization induced by LPS was detected by flow cytometry after SPI1 knockdown. (Q, R) Levels of IL-10 (Q) and IL-1β (R) measured by ELISA after SPI1 knockdown. All experiments were performed in triplicate with independently prepared biological samples. Data are expressed as mean ± SD. * P < 0.05, ** P < 0 .01 compared to the other group.

Article Snippet: ELISA kit for IL-1β (ZC-37974, Zhuocai, China); ELISA kit for IL-10 (ZC-37962, Zhuocai, China); SPI1 antibody (55100-1-AP, Proteintech, China); iNOS antibody (22226-1-AP, Proteintech, China); Arg1 antibody (16001-1-AP, Proteintech, China); Tubulin antibody (80762-1-RR, Proteintech, China); Anti-rabbit IgG (H + L) (14780, Cell Signaling Technology, United States).

Techniques: In Vitro, Flow Cytometry, Knockdown, Control, Enzyme-linked Immunosorbent Assay

Journal: Biochemistry

Article Title: C2 Domain of Protein Kinase C?: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling †

doi: 10.1021/bi026596f

Figure Lengend Snippet: Deduced orientation of the membrane-bound PKCα C2 domain relative to the membrane surface. Shown is a model membrane of pure lipids (44, 45) to which the crystal structure of the isolated PKCα C2 domain has been docked (13). The headgroup layers of the membrane exhibit high heavy atom densities due to the presence crystallographic waters, some of which are highlighted (small space filled molecules), while the central hydrocarbon layer exhibits a lower atom density. For each labeling site on the protein surface, a nitroxide spin label (ball-and-stick, with the nitrogen indicated by sphere) was modeled onto the crystal structure using the program INSIGHT II (Biosym Technologies). Most of the spin labels have been given the g+, g+ dihedral angles believed to be most common (46) except for those where the dihedral angles are constrained to fit the EPR data (M186S1, N189S1, N206S1, R216S1, K230S1, S234S1, R249S1, and R252S1). Also shown are two bound Ca2+ ions (large yellow spheres), the Ca2+ binding loops 1 and 3 that directly contact the membrane, and the phosphate ion (red) bound in the anion binding site on strands 3–4.

Article Snippet: For each labeling site on the protein surface, a nitroxide spin label (ball-and-stick, with the nitrogen indicated by sphere) was modeled onto the crystal structure using the program INSIGHT II (Biosym Technologies).

Techniques: Membrane, Isolation, Labeling, Binding Assay

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