Fig 2: SNX16 is involved in the early stage of IAV replication cycle. (a) HEK293T cells were transfected for 48 h to express empty vector or Flag-SNX16, and then infected with WSN (H1N1) virus at an MOI of 5. At 2 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-Flag pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green). The nuclei were stained with DAPI. (b) A549 cells were transfected with siRNA targeting SNX16 or with scrambled siRNA for 48 h and were then infected with WSN (H1N1) virus at an MOI of 5. At 2 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). The nuclei were stained with DAPI. Data are representative of three independent experiments.

Fig 3: SNX16 is expressed as two obvious bands. HEK293T cells were transfected with plasmids expressing V5- (a) and HA-tagged (b) SNX16, and were subjected to Western blotting with a rabbit anti-V5 pAb and a rabbit anti-HA pAb, respectively. Data are representative of three independent experiments.

Fig 4: Homology analysis of residue R144 of SNX16. The amino acid sequences of SNX16 protein of different species from NCBI were aligned by using DNAMAN software to show homologies of SNX16 (a) and the conservativeness of residue R144 (b).

Fig 5: Downregulation of SNX16 expression promotes the replication of IAV. (a) A549 cells were transfected with siRNA targeting SNX16 or with scrambled siRNA for 48 h and the expression of SNX16 was detected by RT-qPCR (n = 3), ****, p < 0.0001. (b) Viability of A549 cells treated with SNX16-specific or scrambled siRNA was determined by using a CellTiter-Glo assay (n = 3). (c) A549 cells treated with siRNA for 48 h were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells (n = 3). ***, p < 0.001. Data are representative of three independent experiments.