FIG 2.

FFAR2 positively regulates IAV replication in A549 cells. (A to D) FFAR2 siRNA- or scrambled siRNA-transfected A549 cells were infected with AH05 (H5N1) (MOI = 0.1) (A), WSN (H1N1) (MOI = 0.01) (B), SH13 (H9N2) (MOI = 0.1) (C), or VSV-EGFP (100 TCID₅₀) (D) virus. Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells (A to C) or by determining the TCID₅₀ on HEK293T cells (D). **, P < 0.01; ***, P < 0.001. (E) Schematic diagram of four sgRNA targeting sites at the FFAR2 gene loci and the two kinds of FFAR2 truncated mutants. Sanger sequencing results indicated that a frameshift occurred in the FFAR2-truncated mutants within the FFAR2_KO cell line; the knockout of FFAR2 was confirmed by Western blotting. (F) Cell viability of FFAR2_KO A549 cells was measured by using the CellTiter-Glo assay. (G) AH05 (H5N1) virus replication in FFAR2_KO A549 cells. FFAR2_KO A549 cells or control cells were infected with AH05 (H5N1) (MOI = 0.1). Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by means of plaque assays on MDCK cells. ***, P < 0.001. (H) The expression of FFAR2 in FFAR2_KO_Comp A549 cells was confirmed by Western blotting. (I) AH05 (H5N1) virus replication in FFAR2_KO_Comp A549 cells. FFAR2_KO_Comp A549 cells or control A549 cells were infected with AH05 (H5N1) (MOI = 0.1). Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by means of plaque assays on MDCK cells. (J) Cell viability of A549 cells treated with 4-CMTB [2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide] or Cmp58 [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] was measured by using the CellTiter-Glo assay. (K) A549 cells were pretreated with 4-CMTB or Cmp58 at the indicated concentrations for 3 h and then infected with AH05 (H5N1) virus (MOI = 0.1). Supernatants were collected at 24 h p.i., and virus titers were determined by means of plaque assays on MDCK cells. ***, P < 0.001.