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. 2018 Oct 9;115(43):E10069–E10078. doi: 10.1073/pnas.1811892115

Fig. 5.

Fig. 5.

Rapamycin promotes IAV infection in an IFITM3-dependent manner. (A) HeLa were seeded in 24-well plates (50,000 per well) overnight and transfected with siRNA (nontargeting control or a mixture targeting IFITM1, IFITM2, and IFITM3) for 48 h. Cells were treated with DMSO or rapamycin (20 μM) for 4 h and then with fresh media containing DMSO or rapamycin (20 μM) and the indicated quantity of IAV PR8. Approximately 18 h after virus exposure, cells were fixed/permeabilized, stained with anti-NP, and analyzed by flow cytometry. Numbers inside gates indicate percent of NP+ cells. (B) Relative infection scores from A using a multiplicity of infection (MOI) of 0.2 were normalized to DMSO-treated siRNA control cells and averaged. (C) Primary HFFs were seeded in 24-well plates overnight and subsequently transfected, treated, and infected as in A with an MOI of 0.2. (D) MEFs derived from ifitm−/− mice and the same cells transduced with murine IFITM3 (mIFITM3) were seeded in 24-well plates (50,000 per well) overnight. Cells were treated with DMSO or rapamycin (20 μM) for 4 h and then with fresh media containing DMSO or rapamycin (20 μM) and IAV PR8 at an MOI of 0.2. Relative infection scores were normalized and averaged, with infection of DMSO-treated ifitm−/− plus mIFITM3 cells set to 100. (E) Western blot analysis of whole cell lysates derived from ifitm−/− and ifitm−/− plus mIFITM3 MEF treated with DMSO, rapamycin (20 μM), or rapamycin and bafilomycin A1 (1 μM) for 4 h. Immunoblotting was performed with anti-IFITM3. Tubulin served as loading control. (F) HeLa were seeded and transfected as in A and exposed to Sendai virus (Cantell) at an MOI of 0.1. Approximately 18 h after virus exposure, cells were fixed/permeabilized, stained with polyclonal anti-SeV, and analyzed by flow cytometry. Relative infection scores were normalized to DMSO-treated siRNA control and averaged. All error bars indicate SE from three experiments. Statistical analysis was carried out with a Student’s t test. ns, not significant, P > 0.05; *P < 0.05; **P < 0.005. NP, nucleoprotein.