human monocytic leukemia cell line  (ATCC)

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: High‐throughput screening identifies CeeNU as an effective NLRP3 inhibitor. (A and B) The process of selecting the most effective drug CeeNU from the FDA‐approved Drug Library by CellTiter‐Glo. (C) The chemical structure of CeeNU. (D) THP‐1 cells were exposed to graded concentrations of CeeNU for 24 h, and cell viability was subsequently determined by the CCK‐8 assay. (E) LPS‐primed THP‐1 cells were treated with CeeNU for 1 h, stimulated with nigericin for an additional hour, and cell viability was then measured using the CellTiter‐Glo Luminescent Assay. *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: High Throughput Screening Assay, Drug discovery, CCK-8 Assay, Luminescence Assay

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU remarkably represses pyroptosis and IL‐1β release dependent on NLRP3 in human macrophages. (A, B, D, and E) PMA‐differentiated human macrophage THP‐1 cells were either treated with LPS for 3 h plus CeeNU for 1 h, followed by stimulation with nigericin (A and B) or MSU (D and E), the supernatant was collected for ELISA of IL‐1β (A and D) and LDH assay (B and E). (C and F) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant and pro‐IL‐1β, procaspase‐1, NLRP3, GSDMD, GSDMD‐NT, and GAPDH in cell lysates of PMA‐differentiated THP‐1 cells primed with LPS for 4 h in the presence of CeeNU or control, prior to stimulation with nigericin (C) or MSU(F). (G) PMA‐differentiated THP‐1 cells were treated sequentially: first with LPS for 3 h, then with CeeNU for an additional hour. Following this, cells were stimulated with nigericin for 1 h. Subsequently, cells were stained with propidium iodide (PI, 2 µg/mL) to label dying cells and DAPI (1 µg/mL) to label all nuclei for 15 min. Imaging was performed using a 10× objective on an Olympus confocal microscope. Merged images include the corresponding bright‐field view. (H) The number of PI‐positive cells was counted in five randomly chosen microscopic fields. Lytic cell death was defined as the ratio of PI‐positive cells to the total number of DAPI‐positive nuclei and is expressed as a percentage. (I) Scanning electron microscopy of cell surface perforations caused by GSDMD in THP‐1 (Bar = 3 µm). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot, Control, Staining, Imaging, Microscopy, Electron Microscopy

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU is a specific inhibitor of the NLRP3 inflammasome. (A and C) Production of cleaved‐IL‐1β from LPS‐primed BMDMs treated with CeeNU before transfected with poly(dA:dT) (A), flagellin (C). (B and D) LDH release from LPS‐primed BMDMs treated with various doses of CeeNU before transfected with poly(dA:dT) (B), flagellin (D). (E–H) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant (SN) and pro‐IL‐1β, procaspase‐1, NLRP3, and GAPDH in cell lysates of BMDMs (E and F) and THP‐1 (G and H), LPS‐primed BMDMs and THP‐1 cells treated with CeeNU for 1 h before transfected with poly(dA:dT) for 16 h (E and G), flagellin for 16 h (F and H). (I and K) Production of cleaved‐IL‐1β from LPS‐primed THP‐1 cells treated with CeeNU before transfected with poly(dA:dT) (I), flagellin (K). (J and L) LDH release from LPS‐primed THP‐1 cells treated with various doses of CeeNU before transfected with poly(dA:dT) (J), flagellin (L). (M) BMDMs were treated with or without LPS, and plus different concentrations of CeeNU for 4 h, cell lysates were collected for Western blotting. (N) Western blotting analysis of p20 in SNs, pro‐casp1, and NLRP3 in input from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (O) ELISA of IL‐1β in SNs from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (P) LDH release from SN of LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Transfection, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU specifically inhibits NLRP3 inflammasome assembly. (A–D) The treated THP‐1 (A and C) or BMDMs (B and D) cells were incubated with ASC primary antibody overnight, then re‐stained with secondary antibody for 1.5 h, and then stained with DAPI for 10 min before immunofluorescence images were taken by Olympus confocal microscope. Green represents ASC, DAPI (blue) shows nuclei. Five fields were randomly selected for each group of samples. The circular strong fluorescent spot indicated in the figure by the arrow was ASC speck. After that, the average fluorescence intensity was analyzed by ImageJ software, and the data were processed by GraphPad. (E) Western blotting analysis of ASC oligomerization level after cross‐linking with DSS in BMDMs. (F) Western blotting analysis of NLRP3 self‐oligomerization level of BMDMs pretreated with different doses of CeeNU after LPS stimulation for 3 h, then plus with nigericin. (G and H) BMDMs were primed with LPS, treated with 50 µM CeeNU, and then stimulated with 5 mM nigericin for 1 h. The interaction between endogenous NEK7 and NLRP3 was assessed by co‐immunoprecipitation (co‐IP) using an anti‐NEK7 (or anti‐NLRP3) antibody, followed by immunoblotting with antibodies against the reciprocal protein. (I) HEK293T cells were co‐transfected with plasmids expressing GFP‐NLRP3 and FLAG‐NEK7. Following transfection, cells were treated with 50 µM CeeNU or vehicle control as indicated. Cell lysates were subjected to IP using an anti‐GFP or FLAG antibody, followed by immunoblotting with antibodies against FLAG and GFP. ** p < 0.01. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Incubation, Staining, Immunofluorescence, Microscopy, Fluorescence, Software, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Expressing, Control

Journal: iScience

Article Title: Serglycin modulates inflammation and metabolism in macrophages

doi: 10.1016/j.isci.2026.115235

Figure Lengend Snippet: SRGN deficiency disrupts macrophage inflammatory activation and alters mitochondrial function (A) Experimental design. Wild-type ( SRGN +/+ ) and knockout ( SRGN −/− ) THP-1 cells were differentiated into macrophages (M0) and polarized to M1 with LPS and IFN-γ for 24 h, followed by RNA-seq and proteomic analysis of culture supernatants ( n = 4). (B) Relative SRGN expression in wild-type THP-1 cells, showing significant induction after M1 polarization (mean ± SEM; unpaired Student’s t test; ∗∗ p < 0.0001). (C) Gene set enrichment analysis (GSEA) of the macrophage activation signature comparing SRGN −/− and SRGN +/+ THP-1 cells. Knockout cells displayed a reduced extent of activation compared to wild-type cells. (D) Differential expression analysis (volcano plot) of SRGN −/− versus SRGN +/+ THP-1 M1 cells. A total of 2,554 genes were significantly altered (adjusted p < 0.05, fold-change threshold of 1.2), with 1,112 upregulated and 1,442 downregulated in knockout cells. (E and F) Hallmark pathway enrichment analysis of significantly differentially expressed genes. Upregulated pathways in SRGN −/− THP-1 cells included inflammatory response, interferon-γ response, interferon-α response, IL2/STAT5 signaling, and epithelial-mesenchymal transition (E). Downregulated pathways included mTORC1 signaling, glycolysis, cholesterol homeostasis, bile acid metabolism, and reactive oxygen species (ROS) pathways (F). Significant pathways are marked with an asterisk (∗). (G) Overlap of differentially expressed genes between SRGN −/− THP-1 cells and Srgn −/− murine macrophages. A total of 115 commonly upregulated and 189 commonly downregulated genes were identified. (H–I) Pathway enrichment analysis of commonly altered genes. Upregulated genes were enriched for inflammatory response and interferon pathways (H), whereas downregulated genes were enriched for TNF-α/NF-κB signaling and glycolysis (I). (J) Overlap of TNF-α signaling genes between human and mouse macrophages, revealing 15 commonly upregulated and 9 commonly downregulated genes. Notably, commonly downregulated genes included ZFP36 , KLF2 , HES1 , and BHLHE40 , regulators of cytokine production and inflammatory programs.

Article Snippet: THP-1 human monocytic cell line , ATCC , RRID: CVCL_0006.

Techniques: Activation Assay, Knock-Out, RNA Sequencing, Expressing, Quantitative Proteomics

Journal: iScience

Article Title: Serglycin modulates inflammation and metabolism in macrophages

doi: 10.1016/j.isci.2026.115235

Figure Lengend Snippet: SRGN deficiency reshapes the inflammatory secretome and transcriptional programs in human macrophages (A) Volcano plot of differentially secreted proteins between SRGN −/− and wild-type THP-1 macrophages under M1 polarization. Among 1,507 quantified proteins, 53 were significantly altered (adjusted p < 0.05), with serglycin being the most downregulated protein in knockout cells. (B) Validation of selected targets by RT-qPCR. SRGN −/− M1 macrophages showed significantly increased expression of IL6 and TNF and reduced expression of CCL5 compared with wild-type cells (mean ± SEM; unpaired Student’s t test; p < 0.05, ∗ p < 0.01, and ∗∗∗ p < 0.0001; n = 4). (C) ELISA quantification of secreted cytokines in culture supernatants. SRGN −/− macrophages secreted significantly less TNF-α, CCL5, and IL-6 compared with wild-type macrophages ( n = 4), consistent with proteomics and RNA-seq data. (D) Transmission electron microscopy (TEM) images of THP-1 M0 and M1 macrophages. Scale bars, 5 μm. Vesicles were manually annotated and quantified in 10 cells per experimental group. The number of vesicles per cell and the percentage of cellular area occupied by vesicles were significantly reduced in both M0 and M1 SRGN −/− macrophages compared with wild-type macrophages. (E) Phagocytosis assay using fluorescently labeled bioparticles. SRGN −/− macrophages exhibited reduced phagocytic capacity under both M0 and M1 conditions (mean ± SEM; unpaired Student’s t test; p < 0.05 and ∗∗ p < 0.001; n = 6).

Article Snippet: THP-1 human monocytic cell line , ATCC , RRID: CVCL_0006.

Techniques: Knock-Out, Biomarker Discovery, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Transmission Assay, Electron Microscopy, Phagocytosis Assay, Labeling

Journal: iScience

Article Title: Serglycin modulates inflammation and metabolism in macrophages

doi: 10.1016/j.isci.2026.115235

Figure Lengend Snippet: SRGN knockout alters the metabolic profile of macrophages (A and B) Oxygen consumption rate (OCR; maximal respiration) in wild-type (WT) and SRGN −/− (KO) THP-1 macrophages under M0 (A) and M1 (B) conditions, with or without oleic acid (OA) supplementation. Oleic acid significantly increased mitochondrial respiration in SRGN −/− macrophages (mean ± SEM; unpaired Student’s t test; ∗∗ p < 0.001 and ∗∗∗ p < 0.0001; n = 3). (C and D) Extracellular acidification rate (ECAR) in WT (C) and SRGN −/− (D) macrophages under M0 and M1 conditions, with or without oleic acid. Oleic acid did not reverse glycolytic reprogramming in M1 macrophages; however, ECAR was altered in SRGN −/− cells (mean ± SEM; p < 0.05; n = 3). (E and F) Reactive oxygen species (ROS) levels measured by DCFDA fluorescence. ROS levels were significantly reduced in SRGN −/− macrophages compared with WT under both M0 (E) and M1 (F) polarization states (mean ± SEM; ∗ p < 0.01 and ∗∗ p < 0.001; n = 6).

Article Snippet: THP-1 human monocytic cell line , ATCC , RRID: CVCL_0006.

Techniques: Knock-Out, Fluorescence