n alpha  (Chem Impex International)

Journal: Cell research

Article Title: Signal-induced NLRP3 phase separation initiates inflammasome activation.

doi: 10.1038/s41422-025-01096-6

Figure Lengend Snippet: Fig. 1 ZDHHC7-mediated NLRP3 palmitoylation is required for NLRP3 activation. a Enriched genes by the genome-wide CRISPR- Cas9 screening. Genes in NF-κB pathway are shown as green spots, genes known in NLRP3 inflammasome are shown as red spots, and genes (red font) with no known function in NLRP3 pathway are shown as red spots. b NLRP3 activation in WT or ZDHHC7–/– THP-1 cells. Cells were pretreated with 10 ng/mL Pam2CSK4 for 3 h, followed by 4 μM nigericin treatment for another 1 h. c NLRP3 activation in THP-1 cells pretreated with 2-BP or not before immunoblotting. Cells were pretreated with 1 μg/mL LPS for 2 h and then with the 2-BP (2.5, 5, 10 μM) for 1 h, followed by 4 μM nigericin treatment for another 1 h. d NLRP3 activation in the WT, ZDHHC7–/– or ZDHHC7–/– THP-1 cells reconstituted with ZDHHC7- or ZDHHS7-mCherry. ZDHHS7, S-acyltransferase catalytic mutant C160S. Cells were treated as in (b). e LDH analysis in (d). f, g Palmitoylation of Flag-hNLRP3 in HEK293T cells with ZDHHC7 expression was detected by ABE (f) or APE assay (g). h HeLa cells stably expressing Flag-hNLRP3 were treated overnight with 2-BP (100 μM) as indicated. The hNLRP3 palmitoylation level was detected by ABE assay. i Palmitoylation of endogenous NLRP3 in WT or ZDHHC7–/– THP-1 cells detected by ABE assay. j B16 cells expressing Flag-mNLRP3 with indicated combined mutations (Supplementary information, Fig. S3e). The mNLRP3 palmitoylation level was detected by APE assay. k Palmitoylation of Flag- hNLRP3-WT, Flag-hNLRP3-C130S, or Flag-hNLRP3-C261S in HEK293T cells with ZDHHC7 expression was detected by ABE assay. l NLRP3 activation in NLRP3–/– or NLRP3–/– THP-1 cells reconstituted with indicated NLRP3. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin treatment for another 1 h. m mNLRP3 activation in WT or Nlrp3–/– immortalized BMDM (iBMDM) cells reconstituted with indicated Flag-mNLRP3. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 6 μM nigericin treatment for another 1 h. Statistical significance was indicated as follows: ns, not significant, ***P < 0.001.

Article Snippet: Zdhhc7−/− (S-KO-00236) and Abhd13−/− (S-KO-12879) mice were generated by and purchased from Cyagen Biosciences.

Techniques: Activation Assay, Genome Wide, CRISPR, Western Blot, Mutagenesis, Expressing, Stable Transfection

Journal: Cell research

Article Title: Signal-induced NLRP3 phase separation initiates inflammasome activation.

doi: 10.1038/s41422-025-01096-6

Figure Lengend Snippet: Fig. 2 ZDHHC7 is required for NLRP3 inflammasome activation in vivo. a, b mNLRP3 activation in WT or Zdhhc7−/−BMDMs by ATP (a) or nigericin (b) treatment. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 5 mM ATP or 6 μM nigericin treatment for another 1 h. c, d Aim2 (c) or Nlrc4 (d) activation in WT and Zdhhc7−/−BMDMs by poly(dA:dT) transfection or S. Typhimurium infection. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 1 μg/mL poly(dA:dT) transfection for 4 h or S. Typhimurium (MOI = 0.5) infection for 1 h. e, f PI staining (e) and percentage of PI-positive cells (f) in WT and Zdhhc7−/−BMDMs treated as in (a–d). g WT or Zdhhc7–/– mice (n = 5) were intraperitoneally injected with PBS or LPS (22.5 mg/kg) for 12 h. IL-1β and TNF-α in sera were detected by enzyme-linked immunosorbent assay (ELISA) analysis. h WT or Zdhhc7–/– mice (n = 12) were intraperitoneally injected with LPS (22.5 mg/kg); survival was calculated using the Mantel–Cox test. i WT mice (n = 5) were intraperitoneally injected with vehicle or 2-bromopalmitate (2-BP, 50 mg/kg) twice 24 h or 1 h before experiments, then subjected to intraperitoneal injection of PBS or LPS (22.5 mg/kg) for 12 h. IL-1β, TNF-α, and IL-6 in sera were detected by ELISA analysis. j WT mice (n = 12) were intraperitoneally injected with 2-BP and LPS as in (i), and survival was calculated using the Mantel–Cox test. Statistical significance was indicated as follows: ns, not significant, **P < 0.01, ***P < 0.001.

Article Snippet: Zdhhc7−/− (S-KO-00236) and Abhd13−/− (S-KO-12879) mice were generated by and purchased from Cyagen Biosciences.

Techniques: Activation Assay, In Vivo, Transfection, Infection, Staining, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cell research

Article Title: Signal-induced NLRP3 phase separation initiates inflammasome activation.

doi: 10.1038/s41422-025-01096-6

Figure Lengend Snippet: Fig. 3 ZDHHC7-mediated NLRP3 palmitoylation is required for NLRP3 aggregation. a Images of HeLa cells stably expressing mNG-NLRP3 were taken at the indicated times after 8 μM nigericin treatment. Red arrows represent NLRP3 aggregates and white triangles represent vesicles. Scale bars, 4 μm. b Images or statistical histogram of HeLa cells stably expressing Flag-hNLRP3 pretreated with 50 μM 2-BP overnight followed by 8 μM nigericin treatment for another 1 h, and immunostained with anti-Flag antibody. Scale bar, 10 μm. The percentage of cells with NLRP3 condensates was quantified from at least 100 cells (n = 3, mean ± s.d., two-sided t-test). ND not detectable. c Images (left) and colocalization analysis (right) of WT, ZDHHC7−/−, and PYCARD−/−THP-1 cells pretreated with 1 μg/mL LPS for 3 h alone, or followed by 4 μM nigericin treatment for another 1 h, immunostained with anti-hNLRP3 or anti-TGN46 antibody. Scale bar, 10 μm. Quantitative analysis of co- localization along a white line was shown. White arrows, colocalized TGN46 and hNLRP3. Yellow arrows, NLRP3-NEK7-ASC specks. ZDHHC7 and ASC expression was analyzed by western blotting assay using antibodies against ZDHHC7, ASC and GAPDH (right). d HeLa WT, ZDHHC7–/–, and ZDHHC7–/– cells reconstituted with ZDHHC7-mCherry (ZDC7) or ZDHHS7-mCherry (ZDS7) were stimulated with 8 μM nigericin for 1 h, and immunostained with anti-Flag antibody before imaging. e Percentage of cells with hNLRP3 aggregates in (d). f Palmitoylation of Flag-hNLRP3 in WT, ZDHHC7–/–, or ZDHHC7–/– HeLa cells reconstituted with ZDHHC7- or ZDHHS7-mCherry was detected by ABE assay. g Images (left) or percentage of cells with hNLRP3 aggregates (right) in HeLa cells stably expressing indicated Flag-hNLRP3 after nigericin treatment. Cells were treated with 8 μM nigericin for 1 h, immunostained with anti-Flag antibody. h Images (right) or percentage of cells with mNLRP3 aggregates (left) in HeLa cells stably expressing indicated Flag-mNLRP3 mutants after nigericin treatment. Cells were treated with 8 μM nigericin for 1 h, and immunostained with anti-Flag antibody. i Images (left) and statistical diagram (right) of mNG-hNLRP3 aggregates in WT and ABHD13−/−

Article Snippet: Zdhhc7−/− (S-KO-00236) and Abhd13−/− (S-KO-12879) mice were generated by and purchased from Cyagen Biosciences.

Techniques: Stable Transfection, Expressing, Western Blot, Imaging

Journal: Cell research

Article Title: Signal-induced NLRP3 phase separation initiates inflammasome activation.

doi: 10.1038/s41422-025-01096-6

Figure Lengend Snippet: Fig. 5 NLRP3 phase separates upon activation. a Images of HeLa cells stably expressing mNG-hNLRP3-3K/A were taken at the indicated times after 8 μM nigericin treatment. Red and yellow arrows represent the fusion and fission of NLRP3 aggregates, respectively. Scale bar, 2 μm. b FRAP of HeLa cells stably expressing mNG-hNLRP3, treated with nigericin for the indicated time. The white boxes indicate bleached sites. Scale bar, 1 μm. The normalized FRAP rate was shown on the right. s, second; n = 3. c HeLa cells stably expressing APEX-hNLRP3 -EGFP or APEX-hNLRP3-3K/A-EGFP (hN3-3K/A) were stimulated with 8 μM nigericin for 1 h before imaging by the transmission electron microscopy. The white boxes (a, b) indicate magnified regions of right images. Scale bars, as indicated. d In vitro NLRP3 droplet formation after diluting mNG- hNLRP3 protein to the indicated concentrations into a phase-separation buffer at room temperature (RT). e NLRP3 (1 μM) LLPS in low salt (60 mM KCl), high salt (250 mM KCl) or high salt with 1.75% PEG8000. Scale bar, 10 μm. Coomassie blue staining of purified mNG-hNLRP3 protein was shown on the right. f NLRP3 droplets formed as in e. Images of hNLRP3 aggregates before and after photobleaching. Scale bar, 10 μm. g Quantification of FRAP data in f. s, second; n = 5. h Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 with indicated mutants after 40 μg/mL imiquimod treatment for 1 h. Scale bar, 10 μm. i Palmitoylation of the WT, Δ140–152, or C130/261S hNLRP3 in HEK293T cells with ZDHHC7 expression detected by the ABE assay. j NLRP3 activation in NLRP3–/–

Article Snippet: Zdhhc7−/− (S-KO-00236) and Abhd13−/− (S-KO-12879) mice were generated by and purchased from Cyagen Biosciences.

Techniques: Activation Assay, Stable Transfection, Expressing, Imaging, Transmission Assay, Electron Microscopy, In Vitro, Staining

Journal: Cell research

Article Title: Signal-induced NLRP3 phase separation initiates inflammasome activation.

doi: 10.1038/s41422-025-01096-6

Figure Lengend Snippet: Fig. 7 Amphiphilic molecules induce NLRP3 phase separation and activation. a In vitro NLRP3 LLPS as in Fig. 6l with the indicated amphiphilic molecules at different concentrations (7.5% or 10%). Scale bar, 10 μm. b The solubility of NLRP3 in whole cell lysates of HEK293T cells expressing Flag-hNLRP3 in the presence of indicated concentration of amphiphilic di-alcohols including 1,5-PD and 1,2-PD. Precipitated NLRP3 (Pre. NLRP3) was detected by immunoblotting. c The solubility of NLRP3 in cell lysates of WT and ZDHHC7−/−THP-1 cells in the presence of the indicated concentrations of ammonium sulfate (g/100 mL whole cell lysate) was analyzed. Experiment was conducted as in (b). d NLRP3 activation in WT or NLRP3–/– THP-1 cells treated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin for another 1 h, or cells pretreated with 1 μg/mL LPS for 30 min, followed by 1,5-PD (1.2%, 2.5%) or 1,2-PD (0.6%, 1.2%) treatment for another 3 h. e NLRP3 activation in THP-1 cells treated by 4 μM nigericin, 2.5% 1,5-PD or 1.2% 1,2-PD in the presence of different concentrations of KCl (5, 10, 20 mM). f NLRP3 activation in the WT or ZDHHC7–/– THP-1 cells treated by nigericin (4 μM), 1,5-PD (2.5%) or 1,2-PD (1.2%) as in (d). g NLRP3 activation in THP-1 cells treated by nigericin in the presence of 1,5-PD, 1,2-PD or not. Cells were pretreated with 1 μg/mL LPS for 2 h, followed by 0.6% 1,5-PD or 0.3% 1,2-PD treatment for 1 h, and 2 μM nigericin treatment for another 1 h. h Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 with the indicated treatment, 0.5% 1,2-PD or 0.5% 1,5-PD for 4 h, 0.1% HFIP or 20 μM doxorubicin for 3 h, or 5 μM paclitaxel for 4 h. i NLRP3 activation in WT, ZDHHC7–/–, or NLRP3–/– THP-1 cells pretreated with 0.2 μg/mL LPS for 3 h and 2 μM nigericin for another 1 h, or pretreated with 0.2 μg/mL LPS for 30 min and doxorubicin (DOX, 2.5 μM, 5 μM) for another 8 h. j NLRP3 solubility in cell lysate from HEK293T cells expressing Flag-hNLRP3 in the presence of indicated concentration of doxorubicin. Precipitated NLRP3 (Pre. NLRP3) was detected by immunoblotting.

Article Snippet: Zdhhc7−/− (S-KO-00236) and Abhd13−/− (S-KO-12879) mice were generated by and purchased from Cyagen Biosciences.

Techniques: Activation Assay, In Vitro, Solubility, Expressing, Concentration Assay, Western Blot, Stable Transfection

Journal: Future Science OA

Article Title: A phytotherapeutic approach to hinder the resistance against clindamycin by MRSA: in vitro and in silico studies

doi: 10.1080/20565623.2025.2458438

Figure Lengend Snippet: The computed MICs of plant essential oils against the tested MRSA strain and MRSA human clinical isolate are expressed as a volume/volume percentage (% v/v) of oil in MHB with the addition of 0.5% Tween 80 ± standard deviation.

Article Snippet: Pure clindamycin (HPI/2019/QCG/RMA/00236) was generously provided by The Hayat pharmaceutical industry (Jordan).

Techniques: Inhibition

Journal: Future Science OA

Article Title: A phytotherapeutic approach to hinder the resistance against clindamycin by MRSA: in vitro and in silico studies

doi: 10.1080/20565623.2025.2458438

Figure Lengend Snippet: A) Antimicrobial activity of the binary formulas, oil-clindamycin against MRSA ATCC 43300.

Article Snippet: Pure clindamycin (HPI/2019/QCG/RMA/00236) was generously provided by The Hayat pharmaceutical industry (Jordan).

Techniques: Activity Assay