Figure 8Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05). Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (c) Cells were lysed and proteins were digested to peptides with Lys-C and trypsin. Then, succinylated peptides were isolated directly from protease-digested protein extracts by immunoaffinity purification (IAP) using an antibody specific for the succinyl-Lysine motif. Next, the modified peptides were further analyzed by LC-MS/MS. Box plots show the distribution of intensities of the succinylated NP "YLEEHPSAGK87succDPK" peptide, computed as the integrated mass spectrometry peak area of the modified K87 succinyl peptide (n=3). The boxplots whiskers correspond to the minimum and maximum values, while the central band is the median. The first and third quartiles (boxes) are merely the average values between the median and the extrema. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05). (d) Interaction of recombinant of wild-type NP (NP K87) or a K87E mutant protein (NP K87E) with the indicated synthetic polyUC RNAs as measured by fluorescence anisotropy. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05). Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05). Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (g) Plaque-Forming Unit assays determined the production of infectious viral particles in cell supernatants. Data information: Data are represented as the mean ± SEM of 3 biological replicates with (g) 2 technical replicates each. Statistical analysis was performed using the paired t-test (g), (*P < 0.05).