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. 2020 Apr 2;27(5):571–585.e6. doi: 10.1016/j.chembiol.2020.03.004

Figure 7.

Figure 7

Chemical Inhibition of VCP ATPase Activity with ML240 Impairs IFITM3 Turnover, Lysosomal Sorting, and Trafficking

(A) Analysis of IFITM3 and ubiquitination levels upon treatment with ML240. HEK293T cells were transfected with HA-IFITM3 for 12 h, treated with DMSO or ML240 (2.5 μM) for another 12 h, and then lysed for western blot analysis. The red asterisks indicate IFITM3 ubiquitination bands with high molecular weights.

(B) Analysis of IFITM3 turnover upon treatment with ML240. HeLa cells expressing HA-IFITM3 were treated with CHX (25 μg/mL) for the indicated times in the presence of DMSO or ML240 (2.5 μM) and lysed for western blot analysis.

(C) Quantification of IFITM3 levels normalized to tubulin levels shown in (B). Data are represented as mean ± SD, n = 3.

(D) Immunofluorescence analysis of HA-IFITM3 in the presence of bafilomycin A1 or ML240. HeLa cells were transfected with HA-IFITM3 and mCherry-LAMP1, treated with DMSO, bafilomycin A1 (100 ng/mL), or ML240 (2.5 μM) for 12 h, and processed for immunofluorescence with an Alexa Fluor 488-conjugated anti-HA antibody. Scale bars, 10 μm. The right panels show magnified squared regions in the corresponding left panels. White arrows indicate enlarged IFITM3-containing compartments.

(E) Relative percentage of DiD-IAV particles colocalized with IFITM3 at the time of dequenching upon ML240 treatment. HeLa IFITM2/3-KO cells expressing BODIPY-labeled IFITM3 were infected with DiD-IAV particles, acutely treated with ML240 (1 μM), and monitored for DiD dequenching and IFITM3 trafficking by time-lapse imaging. Data are represented as mean ± SD of three independent experiments. ∗∗∗∗p < 0.0001 calculated by Student's t test.

(F) Antiviral activity of IFITM3 in the presence of ML240. HeLa IFITM2/3-KO cells were transfected with HA-IFITM3 and infected with IAV (PR8/H1N1) at an MOI of 2.5 for 2 h. Cells were then treated with ML240 (2.5 μM) for 4 h, fixed, and stained with anti-HA and anti-influenza NP antibodies to measure IFITM3 expression and virus infection, respectively, with flow cytometry. “Low HA” and “High HA” indicate cell populations expressing low and high levels of HA-IFITM3, respectively. Data are represented as mean ± SD of three independent experiments. ∗∗∗p < 0.001, n.s. indicates p > 0.05 calculated by Student's t test.