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. 2019 Oct 15;93(21):e00649-19. doi: 10.1128/JVI.00649-19

FIG 1.

FIG 1

Knockdown of TMPRSS2 expression by PPMO T-ex5 inhibits activation and multicycle replication of IAV but not of IBV in Calu-3 cells. (A) Multicycle replication of Anhui/H7N9 in T-ex5-treated Calu-3 cells. Confluent Calu-3 cells were treated with 25 μM T-ex5 PPMO for 24 h or remained untreated (without [w/o]), inoculated with Anhui/H7N9 at an MOI of 0.001, and further incubated in the absence of T-ex5 for 72 h. Virus titers were determined as PFU per milliliter by a plaque assay of supernatants taken at the indicated time points. Results are mean values ± standard deviations (SD) from two independent experiments. (B) Analysis of HA cleavage in T-ex5-treated Calu-3 cells. Calu-3 cells treated with or without T-ex5 for 24 h were inoculated with Anhui/H7N9 at an MOI of 1 and incubated for 24 h in the absence of T-ex5. Cell lysates were subjected to SDS-PAGE and Western blotting using H7-specific antibodies. HA1 is not detected by the antibody. Beta-actin was used as a loading control. (C) Analysis of TMPRSS2-specific mRNA in Calu-3 cells. Cells were treated with 25 μM T-ex5 for 24 h and then incubated without T-ex5 for 72 h. Total RNA was isolated and analyzed by RT-PCR using primers designed to amplify 1,228 nucleotides of full-length TMPRSS2 mRNA. Full-length and truncated Δex5 PCR products are indicated. (D) Multicycle replication of IBV in Calu-3 cells with or without T-ex5 treatment. Confluent Calu-3 monolayers were treated with 25 μM PPMO T-ex5 or remained untreated. Cells were then inoculated with Malaysia/B or Massachusetts/B at a low MOI of 0.01 and incubated for 72 h in the absence of PPMO T-ex5. At the indicated time points, virus titers were analyzed by a plaque assay. Data are mean values ± SD from three independent experiments. (E) Analysis of HA cleavage in T-ex5-treated Calu-3 cells. Cells incubated with or without T-ex5 as described above were infected with Malaysia/B or Massachusetts/B at an MOI of 1 and incubated without further PPMO treatment for 48 h. Cell lysates were subjected to SDS-PAGE and Western blotting using IBV HA-specific antibodies. Beta-actin was used as a loading control.