Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The inflammasome next door: characterizing pyroptosis induction in HCV-infected and uninfected bystander cells in vitro

doi: 10.3389/fcimb.2025.1603739

Figure Lengend Snippet: Viral entry alone is insufficient to trigger HCV-induced pyroptosis. Huh-7.5 cells were infected with HCV at MOI = 1 or left uninfected. Prior to infection, specific virus aliquots were either heat-inactivated or UV-inactivated. Twenty-four hours prior to staining, cells were treated with LPS/Nigericin as a positive control. (A, D) At 3 dpi cells were fixed using acetone and stained for cleaved caspase-1 (green), HCV core (red), and nuclei were stained with DAPI (blue). Analysis was performed using fluorescence microscopy. Scale bar, 100 μ m. Data are representative of at least three independent experiments. (B, C) At 3 or 4 dpi cells were harvested and stained for cleaved caspase-1 before cells were fixed using the caspase-1 kit fixative. Cells were run on a CytoFLEX flow cytometer, and data were analyzed using Kaluza analysis software. Data are presented as the percent of total cells that were caspase-1 + with standard error. ** p< 0.005, **** p< 0.0001. Data are representative of at least two independent experiments performed in triplicate.

Article Snippet: Following incubation with LPS, Nigericin (sodium salt, InvivoGen, tlrl-nig) was added at a concentration of 16.8 μM and left overnight at 37°C.

Techniques: Infection, Virus, Staining, Positive Control, Fluorescence, Microscopy, Flow Cytometry, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The inflammasome next door: characterizing pyroptosis induction in HCV-infected and uninfected bystander cells in vitro

doi: 10.3389/fcimb.2025.1603739

Figure Lengend Snippet: HCV-induced pyroptosis is dependent on fully infectious virion production in infected Huh-7.5 cells but not in untreated, bystander S29 cells. (A–E) Huh-7.5 or S29 cells were infected with HCV at MOI = 1, left untreated or transfected with RNA encoding for JFH1 T , Δ GDD, JFH-sgr, or mock control. Twenty-four hours prior to staining, cells were treated with LPS/Nigericin as a positive control. (A, D) At 3 dpi cells were fixed using acetone and stained for cleaved caspase-1 (green), HCV core (red), and nuclei were stained with DAPI (blue). Analysis was performed using fluorescence microscopy. Scale bar, 100 μ m. Data are representative of at least three independent experiments. (B, C, E) Huh-7.5 cells (red bars) and (E) S29 cells (yellow bars) were harvested and stained for cleaved caspase-1 prior to fixation, using the caspase-1 kit fixative at (B) 3 or (C, E) 4 dpi. (B, C, E) Cells were run on a CytoFLEX flow cytometer, and data were analyzed using Kaluza analysis software. Data are presented as the percent of total cells that were caspase-1 + with standard error. ** p<0.005, *** p<0.0005, **** p<0.0001. (F) Multiple sequence alignment highlighting the T55I substitution in the RIG-I gene of Huh-7.5 cells that is not in the S29 cells or the human reference sequence ( NG_046918.1 ). (G) Western blotting of cell lysates from Huh-7.5 and S29 cells, with or without HCV infection. Membranes were probed for RIG-I and β -Actin. (A–E, G) Data are representative of at least three independent experiments or two independent experiments performed in triplicate.

Article Snippet: Following incubation with LPS, Nigericin (sodium salt, InvivoGen, tlrl-nig) was added at a concentration of 16.8 μM and left overnight at 37°C.

Techniques: Infection, Transfection, Control, Staining, Positive Control, Fluorescence, Microscopy, Flow Cytometry, Software, Sequencing, Western Blot