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. 2020 Jan 6;94(2):e01707-19. doi: 10.1128/JVI.01707-19

FIG 8.

FIG 8

The FFAR2 downstream effectors β-arrestin1 and AP2B1 promote the replication of IAV. (A) A549 cells were transfected with siRNA targeting β-arrestin1 or β-arrestin2 or with scrambled siRNA for 48 h. The levels of β-arrestin1 or β-arrestin2 mRNA in β-arrestin1 or β-arrestin2 siRNA-treated cells were determined by qRT-PCR in cell lysates and standardized to those in the scrambled siRNA-treated cells. ***, P < 0.001. (B) β-Arrestin1- or β-arrestin2-overexpressing cells that were generated by using retrovirally mediated transduction were transfected with siRNA targeting β-arrestin1 or β-arrestin2 or with scrambled siRNA for 48 h. Whole-cell lysates were collected and subjected to Western blotting with a mouse anti-β-arrestin1 mAb or a mouse anti-β-arrestin2 mAb. (C) Cell viability of β-arrestin1 siRNA- or β-arrestin2 siRNA-treated A549 cells was measured by using the CellTiter-Glo assay. (D) A549 cells were transfected with siRNA targeting β-arrestin1 or β-arrestin2 or with scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus (MOI = 0.1). Supernatants were collected at the indicated time points, and virus titers were determined by means of plaque assays on MDCK cells. **, P < 0.01; ***, P < 0.001. (E) BiFC analysis to determine the interaction between FFAR2-VN and β-arrestin1-VC, AP2B1-VN and β-arrestin1-VC, and FFAR2-VN and AP2B1-VC in HeLa cells. Cotransfection of vector VN and vector VC served as a negative control. (F) A549 cells were transfected with siRNAs targeting different subunits of the AP-2 protein complex for 48 h. The levels of mRNA of the indicated gene in the cell lysates were determined by qRT-PCR and standardized to those in the scrambled siRNA-treated cells. ***, P < 0.001. (G) A549 cells were treated with siRNAs targeting different subunits of the AP-2 protein complex for 48 h, and cell viability was measured by using the CellTiter-Glo assay. (H) A549 cells were transfected with siRNA targeting the indicated gene of the AP-2 protein complex or with scrambled siRNA for 48 h and then infected with AH05 (H5N1) virus (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 < 0.01. (I) AP2B1 siRNA- or scrambled siRNA-treated A549 cells were infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a culture temperature shift to 37°C for 30 min to allow for internalization. The cells were then washed with ice-cold PBS-HCl (pH 1.3) before cell lysis. The amount of internalized virus particles was determined by Western blotting with a rabbit anti-NP pAb. (J) A549 cells were pretreated with 200 μM Barbadin or DMSO for 12 h and then infected with AH05 (H5N1) virus (MOI = 5). Whole-cell lysates were collected at the indicated time points and subjected to Western blotting with a rabbit anti-NP pAb. The band intensities of the Western blots were quantified by using ImageJ software and are expressed as relative NP/GAPDH ratios (I and J).