Journal: Journal of Virology

Article Title: Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner

doi: 10.1128/jvi.01274-25

Figure Lengend Snippet: BCoV entry into HRT-18 cells depends on dynamin. ( A ) The maximum safe concentrations of dynasore were determined using the CCK-8 assay. ( B ) Western blot analysis was used to evaluate the BCoV N protein expression levels, with grayscale analysis performed and presented as a bar graph. ( C ) RT-qPCR was performed to assess the BCoV gene copy numbers. ( D ) TCID 50 assay was used to measure the BCoV viral titers in the cell supernatant. ( E ) IFA was used to detect the number of BCoV-infected cells. Scale bar = 100 µm. ( F ) RT-qPCR was used to evaluate the effect of dynasore on the viral entry. ( G ) RT-qPCR was used to evaluate the effect of dynasore on the viral attachment. ( H ) The siRNA silencing efficiency of dynamin was screened; the effects of dynamin-silenced cells on BCoV infection were assessed by ( I ) Western blot, ( J ) RT-qPCR, ( K ) TCID 50 , and ( L ) IFA. ( M ) RT-qPCR was used to evaluate the effect of dynamin-silenced cells on the viral entry; ( N ) RT-qPCR was used to evaluate the effects of dynamin-targeting siRNA on BCoV attachment. Data are presented as the mean ± SD of three independent experiments (not significant, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The inhibitors used in this study included SSAA09E3 (Cat HY-138102, MedChemExpress), a novel inhibitor that blocks the fusion of the viral membrane with the host cell membrane; CPZ (Cat C0982, Sigma), a clathrin-mediated endocytosis inhibitor; nystatin (Cat 475914, Sigma), a caveolae inhibitor that acts as a sterol-binding agent disrupting caveolae; blebbistatin (Cat 203391, Sigma), an inhibitor of micropinocytosis; dynasore (Cat T1848, TargetMol), a dynamin inhibitor; MβCD (Cat T4072, TargetMol), a cholesterol depletion inhibitor; chloroquine (Cat S6999, Selleck) and NH 4 Cl (Cat A9434, Sigma), a potent inhibitor of V-ATPase and a specific inhibitor of acidification of endosomal vesicles; colchicine (Cat HY-16569, MedChemExpress), which inhibits the polymerization of tubulin; E64d (Cat S7393, Selleck), a cathepsin inhibitor; and camostat (Cat HY-13512, MedChemExpress), a TMPRSS2 inhibitor.

Techniques: CCK-8 Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection

Journal: Medical microbiology and immunology

Article Title: The pathogenic responses elicited during exposure of human intestinal cell line with Giardia duodenalis excretory-secretory products and the potential attributed endocytosis mechanism

doi: 10.1007/s00430-024-00806-y

Figure Lengend Snippet: Unless otherwise specified, Caco-2 cells were exposed to Giardia ESPs for the indicated time periods. (A) Upon exposure of IECs with the amount of ESPs of 20, 40 and 100 μg/mL, the mRNA levels of dynamin, clathrin and caveolin-1 were assessed by qPCR analysis. The comparisons were made with the first group of the graph. (B-D) Cells were exposed to ESPs at the concentration of 20 μg/mL. (B) The expression levels of clathrin and caveolin-1 were determined by western blot analysis. (C) Anti-ESP PAB was used to indicate the bonding between ESPs and IECs after a 3-h exposure (scale bar = 20 μm). (D) Binding between ESPs and clathrin/caveolin-1 was assessed by co-IP analysis. All experiments were repeated four times. Data from triplicate wells from a representative of four independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: We used dynamin inhibitor dynasore (100 μM in use; Abmole, Houston, USA), clathrin inhibitor chlorpromazine (CPZ) (10 μM; Abmole, Houston, USA), and caveolin-1 inhibitor genistein (100 μM; Selleck, Shanghai, China) in inhibition analyses.

Techniques: Activation Assay, Concentration Assay, Expressing, Western Blot, Binding Assay, Co-Immunoprecipitation Assay

Journal: Medical microbiology and immunology

Article Title: The pathogenic responses elicited during exposure of human intestinal cell line with Giardia duodenalis excretory-secretory products and the potential attributed endocytosis mechanism

doi: 10.1007/s00430-024-00806-y

Figure Lengend Snippet: Dynasore was dissolved in 0.1% DMSO for use and shown as “dynasore + DMSO” panel here. Caco-2 cells were exposed to Giardia ESPs at the concentration of 20 μg/mL for 3 h. (A) Dynamin inhibition by dynasore blocked the interactions between ESPs and IECs as assessed by immunofluorescence analysis (scale bar = 20 μm). (B and C) Dynamin inhibition attenuated ESPs-induced cleavage of CASP-3, CASP-9 and PARP and elevation of Bax to Bcl-2 ratio as assessed by western blot and gray value analyses. (C) “Control” was compared to “ESPs”, and “ESPs” was compared to “ESPs + dynasore + DMSO”. (D) Dynamin inhibition suppressed ESPs-induced IEC apoptosis as assessed by AO/EB staining (scale bar = 100 μm). (E) Dynamin inhibition suppressed ESPs-induced upregulation of intracellular NLRP3 and TNF-α and released IL-1β and IL-18 as assessed by western blotting. (F) Dynamin inhibition protected claudin-1, claudin-4, occludin and ZO-1 expressions from being influenced by ESP exposure. (G and H) Dynamin inhibition blocked ESPs-induced elevation of LC3-II to LC3-I ratio and reversed ESPs-induced downregulation of p62 as assessed by western blot and gray value analyses. (H) “Control” was compared to “ESPs”, and “ESPs” was compared to “ESPs + dynasore + DMSO”. All experiments were repeated three times. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: We used dynamin inhibitor dynasore (100 μM in use; Abmole, Houston, USA), clathrin inhibitor chlorpromazine (CPZ) (10 μM; Abmole, Houston, USA), and caveolin-1 inhibitor genistein (100 μM; Selleck, Shanghai, China) in inhibition analyses.

Techniques: Concentration Assay, Inhibition, Immunofluorescence, Western Blot, Control, Staining

Journal: Parasites, Hosts and Diseases

Article Title: Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis -derived secretory products

doi: 10.3347/PHD.24049

Figure Lengend Snippet: Effects of dynamin inhibitor (Dynasore) on IL-8 production in HMC-1 cells stimulated with TvSP or LTB 4 . HMC-1 cells were stimulated for 6 (A) or 16 h (B) with or without TvSP in the presence or absence of Dynasore. The amount of IL-8 in culture supernatant was measured by human IL-8 ELISA. Data are expressed as the mean±SD from 4 independent experiments. Significant differences from the values obtained with cells incubated with DMSO in TvSP- or LTB 4 -treated groups are shown. LTB 4 , Leukotriene B 4 ; HMC-1, Human mast cell line; TvSP, Trichomonas vaginalis -derived secretory products; IL-8, interleukin-8. * P <0.05, ** P <0.01.

Article Snippet: Dynasore (dynamin inhibitor I) was purchased from Calbiochem (Gibbstown, NJ, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay