amiloride  (MedChemExpress)

Journal: Cell Discovery

Article Title: Bergeyella cardium variant induces a unique cytoplasmic vacuolization cell death floatptosis in macrophage

doi: 10.1038/s41421-025-00840-x

Figure Lengend Snippet: a Immunoblot analysis of caspase-3 and cleaved caspase-3 (P17) in WT BMDMs infected with BC or BCV (400 MOI) for the indicated time. b TEM analysis of WT BMDMs infected with BCV (400 MOI) for 30 h (left and middle) and quantification of the percentage of cytoplasmic vacuolization and apoptosis-like cell death within the dead cell population (right). N indicates the cell nucleus. A total of 89 dead cells were analyzed. Scale bars, 1.2 μm for the left and 1.5 μm for the middle. c Microscopic analysis of WT BMDMs infected with BC or BCV (400 MOI) in the presence of amiloride hydrochloride (Amilo, 0.5 μM), z-VAD (25 μM), ferrostatin-1 (Fer-1, 1 mM), necrostatin-1 (Nec-1, 100 μM), necrosulfonamide (NSA, 500 nM), Rapamycin (Rapa, 500 nM), Wortmannin (Wort, 0.2 μM), 3-Methyladenine (3-MA, 5 mM), and pyrrolidinedithiocarbamate ammonium (PDTC, 1 μM) for 12 h. Scale bars, 20 μm. d Immunoblot analysis of caspase-3, cleaved caspase-3 (P17), GSDME, cleaved GSDME (GSDME NT ), p-MLKL, MLKL, p-RIP3, RIP3, p-PI3K, and p-S6 in WT BMDMs infected with BC or BCV (400 MOI) together with various inhibitors in c for 20 h. e LDH analysis of WT BMDMs infected with BC or BCV (400 MOI) in the presence of the indicated inhibitors for 20 h ( n = 3 biologically independent samples). Data are from 3 independent experiments ( e ) or representative of 3 independent experiments with similar results ( a – d ). For e , data represent Mean ± SEM, and two-sided Student’s t -test without multiple-comparisons correction, ** P < 0.01, **** P < 0.0001.

Article Snippet: Inhibitors z-VAD (Calbiochem, 627610), necrostatin-1 (Nec-1; Calbiochem, 480065), necrosulfonamide (NSA; MCE, HY-100573), ferrostatin-1 (Fer-1; MCE, HY-100579), pyrrolidinedithiocarbamate ammonium (PDTC; TargetMoI, T3147), 3-Methyladenine (3-MA; APExBIO Technology, A8353), wortmannin (CST, 9951S), rapamycin (MCE, HY-10219), and amiloride hydrochloride (Alomone labs, A-140) were used to treat BMDMs for 2 h with indicated concentration ahead of bacterial infection.

Techniques: Western Blot, Infection

Journal: Cell Discovery

Article Title: Bergeyella cardium variant induces a unique cytoplasmic vacuolization cell death floatptosis in macrophage

doi: 10.1038/s41421-025-00840-x

Figure Lengend Snippet: a TEM analysis of purified OMVs from BC and BCV . BC and BCV were cultured on Columbia blood agar plates for 96 h, followed by growth in BACTEC™ Lytic media for 16 h. The supernatants of BC and BCV were used for OMV purification and comparative analysis. Scale bars, 0.3 μm. b Quantification of the number of OMVs per field in a ( n = 12 random fields; 3 independent experiments). c Microscopic analysis of WT BMDMs treated with OMVs derived from BCV (100 μg) for the indicated time. Scale bars, 30 μm. d Quantification of vacuole size in the BMDMs in c . The largest vacuole per cell was analyzed, and at least 120 cells were quantified for each group. e Immunoblot analysis of caspase-3 and cleaved caspase-3 (P17) in WT BMDMs treated with OMVs derived from BCV (100 μg) for the indicated time. f Microscopic analysis of WT and Slc9a9 − / − BMDMs treated with OMVs derived from BCV (100 μg) for the indicated time. Scale bars, 30 μm. g Quantification of vacuole size in the BMDMs in f . The largest vacuole per cell was analyzed, and at least 120 cells were quantified for each group. h LDH analysis of WT and Slc9a9 − / − BMDMs treated with OMVs derived from BCV (100 μg) for the indicated time ( n = 4 biologically independent samples). i Immunoblot analysis of caspase-3, cleaved caspase-3 (P17), and SLC9A9 in WT and Slc9a9 − / − BMDMs treated with OMVs derived from BCV (100 μg) for the indicated time. j Microscopic analysis of WT BMDMs treated with OMVs derived from BCV (100 μg) in the presence and absence of amiloride hydrochloride (Amiloride, 0.5 μM) treatment for the indicated time. Scale bars, 30 μm. k Quantification of vacuole size in the BMDMs in j . The largest vacuole per cell was analyzed, and at least 120 cells were quantified for each group. l LDH analysis of WT BMDMs treated with OMVs derived from BCV (100 μg) in the presence and absence of amiloride hydrochloride (Amiloride, 0.5 μM) treatment for the indicated time. m Immunoblot analysis of caspase-3, cleaved caspase-3 (P17), GSDME, and cleaved GSDME (GSDME NT ) in WT BMDMs treated with OMVs derived from BCV (100 μg) in the presence and absence of amiloride hydrochloride (Amiloride, 0.5 μM) treatment for the indicated time. Data are from 3 independent experiments ( h , l ) or representative of 3 independent experiments with similar results ( a – g , i – k , m ). Data represent Mean ± SEM for ( b , d , g , h , k , l ), *** P < 0.001, **** P < 0.0001, by two-sided Student’s t -test without multiple-comparisons correction.

Article Snippet: Inhibitors z-VAD (Calbiochem, 627610), necrostatin-1 (Nec-1; Calbiochem, 480065), necrosulfonamide (NSA; MCE, HY-100573), ferrostatin-1 (Fer-1; MCE, HY-100579), pyrrolidinedithiocarbamate ammonium (PDTC; TargetMoI, T3147), 3-Methyladenine (3-MA; APExBIO Technology, A8353), wortmannin (CST, 9951S), rapamycin (MCE, HY-10219), and amiloride hydrochloride (Alomone labs, A-140) were used to treat BMDMs for 2 h with indicated concentration ahead of bacterial infection.

Techniques: Purification, Cell Culture, Derivative Assay, Western Blot

Journal: Cell Discovery

Article Title: Bergeyella cardium variant induces a unique cytoplasmic vacuolization cell death floatptosis in macrophage

doi: 10.1038/s41421-025-00840-x

Figure Lengend Snippet: a Bacterial killing ability of BC and BCV in BMDMs. WT BMDMs were infected with BC (20 MOI) and BCV (20 MOI) for 2 h, followed by treatment with gentamicin (50 μg/mL) for 1 h. Infected BMDMs were washed, lysed, and cultured on Columbia blood agar plates for 96 h to enumerate intracellular (2 h) bacteria. Washed BMDMs were further cultured in fresh media for 22 h, and the total number of intracellular and extracellular bacteria (22 h) was enumerated after culturing on Columbia blood agar plates for 96 h ( n = 3 biologically independent samples). b , c WT female mice were intranasally infected with 4.0 × 10 8 CFU BC ( n = 12) or BCV ( n = 12), and the body weight change ( b ) and bacterial burden in the lungs on Day 1 after infection were measured ( c ). d TEM analysis of OMVs in bronchoalveolar lavage fluid (500 μL volume per mouse) from BC - and BCV -infected mice in c . Scale bars, 0.3 μm. e Microscopic analysis of cytoplasmic vacuolization in isolated AMs from BC - and BCV -infected mice in c . Scale bars, 20 μm. f H&E staining of lung sections from uninfected and BC - and BCV -infected mice in c . Scale bars, 100 μm. g Disease scores based on inflammation in lung sections in f from uninfected (Uninf, n = 2), BC -infected ( n = 4), and BCV -infected ( n = 4) mice. h Expression of genes encoding IL-1α and IL-6 was analyzed in lung tissues from uninfected (Uninf, n = 3) and BC -infected ( n = 10), and BCV -infected ( n = 10) mice in c . i Bacterial killing ability of BCV in WT and Slc9a9 − / − BMDMs. WT and Slc9a9 − / − BMDMs were infected with BCV (20 MOI) for 2 h, followed by treatment with gentamicin (50 μg/mL) for 1 h. Infected BMDMs were washed, lysed, and cultured on Columbia blood agar plates for 96 h to enumerate intracellular (2 h) bacteria. Washed BMDMs were further cultured in fresh media for 22 h, and the total number of intracellular and extracellular bacteria (22 h) was enumerated after culturing on Columbia blood agar plates for 96 h ( n = 3 biologically independent samples). j , k WT and Slc9a9 − / − female mice were intranasally infected with 4.0 × 10 8 CFU BCV ( n = 8 mice for each group), and the body weight change ( j ) and bacterial burden in the lungs on Day 1 after infection were measured ( k ). l H&E staining of lung sections from BCV -infected mice in k . Scale bars, 100 μm. m Disease scores based on inflammation in lung sections in l from BCV -infected WT and Slc9a9 − / − mice ( n = 5 mice for each group). n ELISA analysis of IL-1α and IL-6 in sera from uninfected and BCV -infected WT and Slc9a9 − / − mice ( n = 5 mice for each group) in k . o WT female mice were intranasally infected with BCV (4.0 × 10 8 CFU), and the bacterial burden in the lungs was measured 24 h after infection. Amiloride indicates that the mice were intravenously injected with amiloride hydrochloride (10 mg/kg) twice, at 0 and 12 h after BCV infection ( n = 7 mice for each group). p H&E staining of lung sections from DMSO- or amiloride hydrochloride-treated mice infected with BCV in o . Scale bars, 100 μm. q Disease scores based on inflammation in the lung sections in p ( n = 3 mice for each group). Data are from 2 independent experiments ( j , k ) or representative of 3 independent experiments with similar results ( a – i , l – q ). Data represent Mean ± SEM for ( a – c , g – k , m – o , q ), two-sided Student’s t -test without multiple-comparisons correction, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Inhibitors z-VAD (Calbiochem, 627610), necrostatin-1 (Nec-1; Calbiochem, 480065), necrosulfonamide (NSA; MCE, HY-100573), ferrostatin-1 (Fer-1; MCE, HY-100579), pyrrolidinedithiocarbamate ammonium (PDTC; TargetMoI, T3147), 3-Methyladenine (3-MA; APExBIO Technology, A8353), wortmannin (CST, 9951S), rapamycin (MCE, HY-10219), and amiloride hydrochloride (Alomone labs, A-140) were used to treat BMDMs for 2 h with indicated concentration ahead of bacterial infection.

Techniques: Infection, Cell Culture, Bacteria, Isolation, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: Condensate nanovaccine adjuvants augment CD8 + T-Cell-dependent antitumor immunity through mtDNA leakage-triggered cGAS-STING axis activation

doi: 10.1038/s41392-025-02447-w

Figure Lengend Snippet: Lysosomal escape, biocompatibility, lymph node targeting, and enhanced humoral immune response to OVA PCD. a OVA PCD representative fluorescence micrographs of cytosolic delivery via lysosomal escape. Confocal microscopy images revealed that after 12 h of treatment with Cy3-labeled OVA PCD (2.5 μg/mL, red) or free OVA protein (2.5 μg/mL, control) in RAW 264.7 cells, the nuclei were stained with Hoechst dye (blue), and the lysosomes were stained with LysoTracker dye (green). OVA PCD did not colocalize with the lysosomes, indicating successful escape from the lysosomes into the cytoplasm. Scale bar: 20 μm. b Analysis of the cellular uptake pathways of OVA PCD. Ethamilamiline (EIPA), genistein (GEN), and chlorpromazine (CPZ) were used to inhibit macropinocytosis, caveolin-mediated endocytosis, and clathrin-mediated endocytosis, respectively ( n = 5). c Cytotoxicity of OVA PCD at different concentrations (10, 30, 50, 70, and 100 μg/mL) was assessed via a CCK-8 assay after coincubation with 293 T cells for 24 h (n = 3). d – f C57BL/6 mice were subcutaneously injected at the tail base with 50 µg of Cy5.5-labeled OVA-PCD (equivalent to the same amount of free OVA, n = 3). Representative images of in vivo fluorescence signals in the draining inguinal lymph nodes (LNs) were acquired via the FX Pro small animal imaging system at 12 and 24 h post-injection ( d ). LNs were harvested at the corresponding time points for ex vivo fluorescence signal acquisition ( e ), scale bar: 0.5 cm, and considering the leftward bias in the distribution of the fluorescence signal, a quantitative analysis of the relative fluorescence intensity was performed on the left lymph nodes ( f ). g Timeline of the in vivo humoral immune study of OVA PCD. h Total antibody titers in the serum of BALB/c mice after immunization with different formulations twice (immunized every two weeks, n = 5). i Detection of different IgG subtype levels in the serum of BALB/c mice after immunization with different formulations twice (immunized every two weeks) following serum dilution ( n = 5). All data are expressed as mean ± s.d. from two independent experiments. Unpaired two-tailed Student’s t tests were used for ( c , f ). One-way ANOVA with Tukey’s post hoc test was employed for ( b , h , i ) Significance levels are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant)

Article Snippet: 5-(N-ethyl-N-isopropyl) amiloride (EIPA), chlorpromazine (CPZ) and genistein were purchased from MCE.

Techniques: Fluorescence, Confocal Microscopy, Labeling, Control, Staining, CCK-8 Assay, Injection, In Vivo, Imaging, Ex Vivo, Two Tailed Test