Journal: bioRxiv

Article Title: Bergeyella cardium variant induces unique cytoplasmic vacuolization cell death

doi: 10.1101/2024.09.24.614850

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 times. B , TEM analysis of WT BMDMs infected with BCV (400 MOI) for 30 hours (left and middle) and quantification of the percentage of floatptosis and apoptosis within the dead cell population (right). N indicates the cell nucleus. A total of 89 dead cells were analysed. 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 (Amiloride, 0.5 μM), z-VAD (25 μM), ferrostatin-1 (Fer-1, 1 mM), necrostatin-1 (Nec-1, 100 μM), necrosulfonamide (NSA, 500 nM), Rapamycin (500 nM), Wortmannin (0.2 μM), 3-Methyladenine (3-MA, 5 mM), and pyrrolidinedithiocarbamate ammonium (PDTC, 1 μM) for 12 hours. Scale bars, 20 μm. D , Immunoblot analysis of caspase-3, cleaved caspase-3 (P17), 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 12 hours. E , LDH analysis of WT BMDMs infected with BC or BCV (400 MOI) in the presence of the indicated inhibitors for 12 hours (n=3 biologically independent samples). Data are from 3 independent experiments ( E ) or representative of 3 independent experiments with similar results ( A - D ). Data represent Mean ± SEM for ( E ), 2-sided Student’s t test without multiple-comparisons correction, **P < 0.01, ***P < 0.001.

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 hours with indicated concentration ahead of bacterial infection.

Techniques: Western Blot, Infection

Journal: bioRxiv

Article Title: Bergeyella cardium variant induces unique cytoplasmic vacuolization cell death

doi: 10.1101/2024.09.24.614850

Figure Lengend Snippet: A , Bacterial killing ability of BC and BCV in BMDMs. WT BMDMs were infected with BC (10 MOI) or BCV (10 MOI) for 2 hours. Infected BMDMs were washed, lysed and cultured on Columbia blood agar plates for 96 hours for enumeration of intracellular (2 h cell) bacteria. Washed BMDMs were further cultured in fresh media for 22 hours, and the numbers of intracellular (22 h cell) and extracellular (22 h Sup) bacteria were enumerated after they were cultured on Columbia blood agar plates for 96 hours (n=3 biologically independent samples). B , Growth analysis of BC and BCV in BMDM culture media. ‘0 h’ indicates the starting point of BC and BCV , and ‘24 h’ indicates the number of BC and BCV in the media after 24 hours of growth (n=3 biologically independent samples). C , Microscopic analysis of cytoplasmic vacuolization in Alveolar macrophages (AMs) infected with BC or BCV (400 MOI) for 12 hours. Scale bars, 20 μm. D , E , WT mice were intranasally infected with 4.0×10 CFU BC (n=12) or BCV (n=12), and the body weight change ( D ) and bacterial burden in the lungs on Day 1 after infection were measured ( E ). F , Microscopic analysis of cytoplasmic vacuolization in isolated AMs from BC - and BCV -infected mice in ( E ). Scale bars, 20 μm. G , H&E staining of lung sections from uninfected and BC - and BCV -infected mice in ( E ). Scale bars, 100 μm. H , Disease scores on the basis of inflammation in lung sections in ( G ) from uninfected (Uninf, n=2), BC -infected (n=4), and BCV -infected (n=4) mice. I , Expression of genes encoding IL-1α and IL-6 was analysed in lung tissues from uninfected (Uninf, n=3) and BC -infected (n=10), and BCV -infected (n=10) mice in ( E ). J , WT mice were intranasally infected with BCV (4.0×10 CFU), and the bacterial burden in the lungs was measured at 24 hours after infection. Amiloride indicates that the mice were intravenously injected with amiloride hydrochloride (10 mg/kg) twice, at 0 and 12 hours after BCV infection (n=7 mice for each group). K , H&E staining of lung sections from DMSO- or amiloride hydrochloride-treated mice infected with BCV in ( K ). Scale bars, 100 μm. L , Disease scores on the basis of inflammation in the lung sections in ( K ) (n=3 mice for each group). Data are from 3 independent experiments ( A , B ) or representative of 3 independent experiments with similar results ( C - K ). Data represent Mean ± SEM for ( A , B , D , E , H , I , J , L ), 2-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 hours with indicated concentration ahead of bacterial infection.

Techniques: Infection, Cell Culture, Bacteria, Isolation, Staining, Expressing, Injection