Fig 2: RPS16 promotes IAV replication. A549 cell was infection with influenza A/wsn/1933 virus (MOI=1) for 0, 6, 12 and 24h; (A) the expression of RPS16 mRNA was detected by real-time qPCR and normalized to expression of GAPDH. Data are the mean ± SD from three independent experiments, ****P < 0.0001 vs. 0h control cell by t-test; (B) the expression of RPS16 protein after IAV infection was detected by western blot. Data are representative of at least three independent experiments. (C and D) A549 cells were transfected with siNC or siRPS16. After 48 h, the cells were infected with influenza A/wsn/1933 virus of 0.1MOI, the expression of influenza NP protein was detected by westen blot (C); (D) The virus titer in the supernatants was infected to MDCK cell and measured the viral titer by plaque forming assay, data were shown as mean ± SD (**p < 0.01, ***p < 0.001). (E and F) BEAS-2B cells were transfected with siNC or siRPS16. After 48 h, the cells were infected with influenza A/wsn/1933 virus (MOI = 0.1), the expression of influenza NP protein was detected by westen blot (E). The virus in the supernatants was infected to MDCK cell and measured the viral titer by plaque forming assay (F), data were shown as mean ± SD (**p < 0.01, ***p < 0.001). (G, H) A549 cells were transfected with RPS16 expressing vector or control vector. After 48 h, the transfected cells were infected with influenza A/wsn/1933 virus of 0.1 MOI and 0.5 MOI, the expression of influenza NP protein was detected by westen blot (E); the virus in the supernatants infected with 0.1 MOI was measured the viral titer by plaque forming assay (F). Data were shown as mean ± SD (**p < 0.01, ***p < 0.001). Data are the representative of three independent experiments.

Fig 3: RPS16 is a direct target of let-7. (A) Schematic representation of predicted target sites of hsa-let-7b-3p in the 3’-UTR of RPS16 and mutant RPS16 3’-UTR reporter constructs. (B) 293T cells were co-transfected with RPS16-3’-UTR luciferase reporter plasmid, RPS16-3’-UTR mut luciferase reporter plasmid (100ng/well), together with negative mimic (miR NC) or let-7b-3p/let-7f-3p mimic (50 nM). After 48 h, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are the mean ± SD from four independent experiments. (****P < 0.0001 vs. negative mimic by t-test. (C) RNA pulldown assay indicated that biotin−hsa-let-7b-3p precipitated with RPS16 mRNA, Lac Z(NC)served as a negative control, the precipitated mRNA was reverse transcripted into cDNA and detected by RT-PCR, the relative expression level was compared to the gene expression in the input RNA. Data were shown as mean ± SD, *p < 0.05, ***p < 0.001. (D) A549 cells were transfected with let-7b-3p/let-7f-3p mimic or negative mimic (miR NC) at a final concentration of 10 nM and 50 nM, respectively. After 24 h, the expression of RPS16 was measured by real-time qPCR and normalized to expression of GAPDH. Data are the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 vs. negative control by t-test. (E) A549 cells were transfected with let-7b-3p/let-7f-3p mimic or negative mimic (miR NC) at a final concentration of 10 and 50 nM, respectively. After 48 h, RPS16 protein expression was analyzed by Western blot and the gray scale quantitation of the bands were analyzed by Image J software and normalized to the loading control. β-actin was used as loading control. Data are representative of three independent experiments, *p < 0.05.