Fig 2: The nucleoporin TPR (but not NUP153 and NUP50) is required for influenza A virus mRNA nuclear export.a DLD-1 cells expressing endogenously AID-tagged basket nucleoporins (NUP153AID, NUP50AID and AIDTPR cells) were infected with influenza A virus (A/WSN/33) and after 2 h 45 min cells were untreated or treated with auxin to degrade AID-tagged proteins. After 8 h of infection, cells were fixed and subjected to smRNA FISH to detect viral M, NS, and HA mRNAs with probes labeled with Quasar 570. DNA was stained with Hoechst. b–d NUP153AID cells were infected and treated with auxin as described in a. Viral mRNAs and DNA were detected as in (a). Scale bar, 10 μm. Data are representative of three independent experiments. e–g Fluorescence intensity of each viral mRNA was quantified in the whole cell and in the nucleus using Imaris software (Bitplane). Percentage of nuclear values for each viral mRNA are shown for each cell (dot) (e Untreated n = 20 cells and auxin treated n = 19 cells, f Untreated n = 24 cells and auxin treated n = 25 cells, g Untreated n = 22 cells and auxin treated n = 22 cells). For each condition, cells used for quantification were derived from three independent experiments. h Western blot was performed using anti-NUP153 antibody to confirm NUP153 degradation. i–k NUP50AID cells were infected and treated with auxin as described in (a). Viral mRNAs and DNA were detected as in a. Scale bar, 10 μm. Data are representative of three independent experiments. l–n Quantification of fluorescence intensity was performed as in e (l Untreated n = 19 cells and auxin treated n = 21 cells, m Untreated n = 21 cells and treated auxin n = 20 cells, n Untreated n = 22 cells and auxin treated n = 22 cells). For each condition, cells used for quantification were derived from three independent experiments. o Western blot was performed using anti-NUP50 antibody to confirm NUP50 degradation. p–r DLD-1 and AIDTPR cells were infected and untreated or treated with auxin as described in a. Viral mRNAs and DNA were detected as in (a). Scale bar, 10 μm. Data are representative of three independent experiments. s–u Quantification of fluorescence intensity was performed as in e (s DLD-1 n = 26 cells, untreated n = 24 cells and auxin treated n = 20 cells, t DLD-1 n = 28 cells, untreated n = 27 cells and auxin treated n = 27 cells, u DLD-1 n = 27 cells, untreated n = 27 cells and auxin treated n = 26 cells). For each condition, cells used for quantification were derived from three independent experiments. v Western blot was performed using anti-TPR antibody to confirm TPR degradation. Graphs show mean ± SD. p values were calculated using unpaired two tailed Student’s t test (GraphPad Prism 9). p values ≤0.05 are considered significant.