Fig 5.

NSC95397 modulates NS1 nuclear localization and its association with cellular chromatin. (A and B) BEAS2B cells were infected with influenza A/WSN/33 virus at MOI = 1 in the presence of DMSO or NSC95397 and fixed or harvested at 4 or 7 hpi. (A) Fixed cells were stained for NS1 (red), NP (green), and nuclei (DAPI; blue). White arrowheads signify nuclear NS1 staining. (B) Protein extracts from 7 hpi were subjected to subcellular fractionation. Total cell lysates and the chromatin-bound protein fraction were used for immunoblot analyses using antibodies against viral NS1 and NP proteins and using cellular GAPDH, histone H3, and transcription factor SP1 as fractionation controls. At 7 hpi, viral NS1 and NP was found associated with cellular chromatin in DMSO-treated but not in NSC95397-treated cells. (C) BEAS2B cells were infected with wild-type (wt) or reconstructed influenza A/WSN/33 virus expressing NS1 R38AK41A (NS1 RK) in the presence of DMSO or 2 μM NSC95397 at MOI = 0.1. Culture supernatants were collected for virus titration by a plaque assay at 24 hpi, and the results are presented as percent inhibition compared to DMSO-treated cells for each respective virus, set as 100%. n.s., not significant; ***, P < 0.001.