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

Journal: Cell reports

Article Title: Immune gene diversity and STING1 variants in shaping cancer immunity across different genetic ancestry populations

doi: 10.1016/j.celrep.2025.116882

Figure Lengend Snippet: (A) The K-means clustering algorithm was used to group the cancer cell lines based on their similarity of IFN-β and ISG expression in response to cGAMP treatment (10 μg/mL, 30 min). The STING1 haplotypes of each cell line are indicated using color coding. (B) Heatmap plot illustrates the changes in expression levels of IFN-β and ISGs following cGAMP treatment. The K-means group and STING1 haplotypes of each cancer cell line are represented by color codes at the top of the heatmap. (C) Illustration of the dual gRNA/CRISPR strategy used to knock out an entire STING1 gene allele in the STING1 heterozygous cancer cell lines. (D) rhPCR-based allelic discrimination was used for genotyping the STING1 gene in the collected clones. The normalized reporter signals (Rn) for allele 1 (FAM) and allele 2 (VIC) were plotted on the x and y axes, respectively. The allelic discrimination plot shows three distinct genotype clusters, including individuals homozygous (depicted in red) and heterozygous (shown in green) for the reference allele and those homozygous for the alternate allele (represented in blue). The homogeneous HAQ/HAQ line HARA and WT/WT line H1373 served as genotyping controls. (E) Western blot analysis of p-TBK1 and p-STAT1 was conducted in isogenic SKOV3 cells carrying either WT or HAQ STING1 , following treatment with 10 μg/mL cGAMP. (F) Bubble plot illustrates the results of gene set enrichment analysis (GSEA) for the upregulated genes identified by RNA-seq following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . The IFN gene sets were obtained from Reactome, and the transcription-factor-regulated gene sets were sourced from TRANSFAC. The bubble size corresponds to the −log (adjusted p value), and the color intensity indicates the number of genes within each gene set. (G) Violin plot shows the fold changes of the commonly upregulated genes identified by RNA-seq after treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . (H) The fold changes in expression levels of IFN-β and ISGs, as measured by RT-qPCR, following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 and TOV21G cells carrying WT/HAQ, WT, or HAQ STING1 . (I) The numbers of T cells (Jurkat-CXCR3, top) and NK cells (NK92, bottom) that migrated to the lower compartment, where isogenic SKOV3 cells were pretreated with 5 μg/mL poly(dA:dT) for 4 h. (J) WT or HAQ STING1 cDNA was transduced into SKOV3 or TOV21G cells in which the endogenous STING1 had been knocked out using CRISPR. The expression of STING1 protein was subsequently detected through western blots. (K) The levels of IFN-β expression measured by ELISA following treatment with 10 μg/mL cGAMP for 30 min in SKOV3 and TOV21G cells transduced with either WT or HAQ STING1 . (L) WT or HAQ STING1 cDNA was transduced into HEK293 reporter cells, which lacked endogenous STING1 expression and contained dual reporters. The expression of STING1 protein was subsequently detected through western blots. (M) The activities of ISRE and hIFN-β reporters after treatment with 10 μg/mL cGAMP for 30 min in 293-Dual Null reporter transduced with either WT or HAQ STING1 . Data are represented as means ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: 2’3’-cGAMP (Cat No: tlrl-nacga23–1) and poly (dA:dT) (Cat No: tlrl-patn-1) were procured from Invivogen and dissolved in sterile, endotoxin-free water.

Techniques: Expressing, CRISPR, Knock-Out, RNase H-dependent PCR, Clone Assay, Western Blot, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transduction