huh7 5 cells  (Thermo Fisher)

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: Translational activity in virus-infected cells was determined using RPM assays. Naïve Huh7 cells, NaAs-treated cells (50 μM for 30 min) or cells infected with ZIKV, YFV/17D or YFV/Asibi at MOI 1 for 48 h were incubated with 10 μg/ml of puromycin to label nascent peptide chains prior to fixation. Puromycin-labelled peptides were visualized via immunostaining against puromycin (green), infected cells were detected by staining for the flavivirus envelope protein (red) and SGs were observed by staining for eIF3η (yellow). Nuclei were stained with DAPI. A) Representative images for the indicated conditions at 48 hpi. Scale bars 10μm. B) Quantification of the cell fraction displaying SGs in the conditions indicated at 24 (filled circle) or 48 hpi (open circle). SG formation was assessed by visually inspecting single cells for eIF3η-postive granules. Mock and NaAs-treated cells were included as negative and positive controls respectively. For virus- infected conditions, only cells positive for the flavivirus envelope protein were considered. For each condition at least 170 cells were quantified from at least three biological replicates. Statistical significances relative to mock are indicated at the top. C) and D) Quantification of translational shut-off for representative replicates for Huh7 cells infected with C) ZIKV and YFV/17D or D) YFV/Asibi at 48 hpi. Mock and NaAs-treated cells were included as negative and positive controls respectively. Single cells were manually measured for the puromycin signal intensity. For virus-infected conditions, only cells positive for the flavivirus envelope protein were considered. a.u. arbitrary units. Statistical significances and the number of analysed cells (n) are given at the top.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Activity Assay, Virus, Infection, Incubation, Immunostaining, Staining

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: YFV-induced SGs are sensitive to CHX-mediated disassembly. Huh7 cells infected with ZIKV, YFV/17D or YFV/Asibi at MOI 1 for 48 h were treated with 20 μM CHX for 30 min or left untreated (ctr.) prior to fixation. Silvestrol-treated cells (1 μM for 30 min) were included as positive control for SG formation and CHX-sensitivity. The presence of SGs was visualized via immunostaining against G3BP1 (green), and infected cells were detected by staining for the flavivirus envelope protein (red). Nuclei were stained with DAPI. A) Representative images for the indicated conditions at 48 hpi. Scale bars 10μm. B-D) SG formation was assessed by visually inspecting single cells for G3BP1-postive granules. For virus- infected conditions, only cells positive for the flavivirus envelope protein were considered. The number of cells analysed (n) from three biological replicates and statistical significances are indicated at the top. B) Quantification of the cell fraction displaying SGs. C) Measurement of the average SG size in cells displaying SGs. SG size is given in microns. D) Quantification of the SG number in cells displaying SGs.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Infection, Positive Control, Immunostaining, Staining, Virus

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: A) and B) Huh7 cells were transfected with plasmids expressing the indicated flag-tagged YFV/Asibi viral proteins or left untransfected (UT) for 48 h. 30 min prior to fixation cells were treated with 50 μM NaAs for 30 min. Transfected cells were identified by immunostaining for the flag tag and the fraction of transfected cells displaying SGs was assessed by staining for G3BP1. A) Representative images for the indicated conditions. Scale bars 10μm. B) Quantification of Huh7 cells expressing the transfected protein and displaying SGs. Depicted are three biological replicates with the statistical significances given at the top. C) Upper panel: Table depicting peptides used to measure NTF2L binding affinity in fluorescence anisotropy binding experiment. YFV-C38-62 contains two overlapping predicted NTF2L-binding motifs (underlined), YFV-C47-51 and YFV-C49-53; F49A, F51A, and F49/51A are mutations that disrupt the binding determinants of the predicted NTF2L-binding motifs (highlighted in bold and red); ZIKA-C43-64 and JEV-C41-62 are located at corresponding regions as the NTF2L-binding site in YFV-C. SFV-nsp3 is a known NTF2L binding peptide. Lower panel: the binding affinity (dissociation constant; Kd) of each peptide in upper panel to the NTF2L domain, measured via fluorescence anisotropy binding experiment.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Transfection, Expressing, Immunostaining, FLAG-tag, Staining, Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: To prove that the G3BP1-inhibitor G3Ib leads to the disassembly of virus-induced SGs, Huh7 cells infected with ZIKV, YFV/17D or YFV/Asibi at MOI 1 for 48 h were treated with 50 μM G3Ib or the inactive enantiomer G3Ib’ for 30 min prior to fixation. Silvestrol-treated (1 μM for 30 min) Huh7 cells were included as a control for G3Ib-mediated SG disassembly. The presence of SGs was visualized via immunostaining against G3BP1 (green), and infected cells were detected by staining for the flavivirus envelope protein (red). Nuclei were stained with DAPI. A) Representative images for the indicated conditions at 48 hpi. Scale bars 10μm. B-C) SG formation was assessed by visually inspecting single cells for G3BP1- postive granules. For virus-infected conditions, only cells positive for the flavivirus envelope protein were considered. The number of cells analysed (n) from three biological replicates and statistical significances are indicated at the top. B) Quantification of the cell fraction displaying SGs. C) Quantification of the SG number in cells displaying SGs. D-E) Huh7 cells were infected with ZIKV, YFV/17D or YFV/Asibi at MOI 1 for 48 h in the presence of 50 μM G3Ib or the inactive compound G3Ib’ or DMSO as controls. D) Cells were harvested for RNA isolation and subsequent RT-qPCR. Ct values for the viral genome were normalized to the Ct value for the housekeeping gene tubulin (-dCT). E) Supernatant from infected cells was harvested and viral titer determined via plaque assay. Depicted are the log10-transformed PFU/ml. Statistical significances from three biological replicates are indicated at the top.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Virus, Infection, Control, Immunostaining, Staining, Isolation, Quantitative RT-PCR, Plaque Assay, Transformation Assay

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: A) and B) GFP-G3BP1 expressing Huh7 cells were either treated with NaAs (0.5 mM for 1 h), infected with YFV/Asibi at MOI 20 or left untreated (mock). At 24 hpi cells were harvested and enriched for the SG-containing fraction, followed by immunoprecipitation for GFP-G3BP1. The resulting eluate was analysed by mass spectrometry. Only proteins for which at least two peptides were found in at least 2 out of 3 replicates were selected. A) Venn diagram displaying the number of candidates identified in the indicated conditions . B) GO pathway enrichment analysis using WebGestalt of the 115 proteins that were exclusively present in YFV/Asibi-induced SGs. Displayed are most significant terms found in each subset of proteins for biological processes (BP), cellular compartment (CC), comprehensive resource of mammalian protein complexes (CORUM) and molecular function (MF). C) siRNA screen of 62 candidates exclusively found in YFV/Asibi-induced SGs. Huh7 cells were transfected with four siRNAs targeting the respective candidate. At 48 h post transfection cells were infected with YFV/Asibi at MOI 20 for 24 h. Infectivity was determined using flow cytometric analysis of cells stained for the viral envelope protein. The percentage of infected cells transfected with a non-target (NT) control is indicated as a grey area. Statistical significances compared to the NT control are indicated at the top.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Expressing, Infection, Immunoprecipitation, Mass Spectrometry, Transfection, Staining, Control

Journal: bioRxiv

Article Title: Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

doi: 10.1101/2025.11.13.688208

Figure Lengend Snippet: A) and B) Huh7 cells that stably express a mitochondria-targeted fluorophore (mtTq) were infected with ZIKV, YFV/17D or YFV/Asibi at MOI 1 for 48 h. As positive control for mitochondrial fragmentation cells were treated with 50 μM CCCP for 2 hours prior to fixation. Mitochondria are depicted in turquoise; infected cells were detected by immunostaining for the flavivirus envelope protein (red). A) Representative images for the indicated conditions at 48 hpi. Scale bars 10μm. Upper panel, overview; lower panel, zoom in. B) Quantification of Huh7 cells displaying the indicated mitochondrial phenotypes. For each condition at least 120 cells were quantified from a total of three biological replicates. C) ROS levels were determined in naïve Huh7 cells or cells infected with ZIKV, YFV/17D or YFV/Asibi at MOI 2 for 48 h. As control for ROS production cells were treated with 50 μM CCCP for 2 h prior analysis. To measure ROS levels cells were incubated with MitoSoxRed reagent for 30 min prior to determination of the average red object mean intensity by the IncuCyte S3 live cell analysis software. Depicted are four biological replicates with their significances at the top. D) Analysis of the mitochondrial membrane potential (MMP) from cells infected with ZIKV, YFV/17D or YFV/Asibi at MOI 2. MMP was monitored for four days every hour using the MMP orange reagent kit and the IncuCyte S3 live cell analysis software. The red mean intensity object average was normalized to 3 hpi. SDs for each time point of five biological replicates are indicated with the shaded area. MMP curves were compared to mock samples using two-way ANOVA with Šídák’s multiple comparisons test. hpi, hours post-infection. E) Analysis of de novo mitochondrial protein translation. Naïve Huh7 cells, CCCP-treated cells (50 μM for 2 h) or cells infected with YFV/17D or YFV/Asibi at MOI 1 for 48 h were incubated with 50 μg/ml CHX and 1mM HPG to label nascent peptide chains within mitochondria. Upon fixation, HPG was chemoselectively ligated to an azide-containing Alexa Fluor 555 dye. Single cells were manually measured for the HPG signal intensity. Depicted is a representative replicate out of five replicates. a.u. arbitrary units. Statistical significances and the number of analysed cells (n) are given at the top.

Article Snippet: Huh7 mito- mTurquoise2 cells (kindly provided by A.Ruggieri, CIID Heidelberg) were cultured under the addition of 200 ug/ml zeocin (InvivoGen, #ant-zn-1).

Techniques: Stable Transfection, Infection, Positive Control, Immunostaining, Control, Incubation, Cell Analysis, Software, Membrane