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. Author manuscript; available in PMC: 2020 Apr 20.
Published in final edited form as: Cell Host Microbe. 2018 Mar 14;23(3):382–394.e5. doi: 10.1016/j.chom.2018.02.005

Figure 6. Activated EGFR Is Associated with DID-HCV at the Tight Junction prior to Internalization.

Figure 6.

(A–E) Huh-7.5 organoids were infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for the indicated times, fixed, and probed for either total EGFR or phosphoEGFR 1045. (A, B, and D) Arrows indicate area enlarged in insets; dashed arrows indicate DiD colocalization while solid arrows denote DiD-HCV particles lacking colocalization. (A) DiD-HCV colocalization over a time course of infection. (B) DiD-HCV colocalization with phospho-EGFR at 120 min post shift. Lower inset is merged and enlarged in upper inset. (D) DiD-HCV colocalization at 0 min post shift, analyzed for localization of DiD-HCV particles. (C and E) Quantification of (A) and (C), respectively. (F) Huh-7.5 organoids were serum starved, infected with concentrated HCV for 1 hr at 4C, shifted to 37°C, processed with Matrigel cell recovery solution, and lysed at the indicated times. Lysate samples were immunoblotted for the specified proteins. (G) Wild-type, shEGFR, and shEGFR cells expressing wild-type or mutant EGFR were seeded into 6-well plates, lysed, and analyzed via immunoblot with indicated antibodies. (H) Wild-type, shEGFR, or complemented cells were seeded onto 96-well plates, infected with HCV for 48 hr, and then analyzed for relative HCV RNA levels. n = total DiD signal, mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.