Figure 5.

In the Context of Reovirus Infection, GSK2606414 Promotes eIF2a-ATF4 Signaling via PERK or GCN2 with Increased Viral Protein Levels ATF4 Dependent

(A) Western blot of cell lysates from 2D culture validating Tet-pLKO shRNA knockdown of PERK, GCN2, and PKR after 96 h of doxycycline treatment. (B) FaDu and (C) HN5 cells were co-infected with ATF4-mCherry reporter in combination with Tet-pLKO SCR, PERK, GCN2, or PKR targeting shRNA. Spheroids were treated for 96 h with doxycycline before treatment with GSK2606414 and reovirus. Reporter expression was imaged at 72 h, matching the time point used in Figure 4. Control values (black circles) are for parental Tet-pLKO cell lines in the absence of doxycycline knockdown, and shRNA values (blue squares) correspond to doxycycline-induced shRNA expression and knockdown. (D) FaDu and HN5 cells were infected with Tet-pLKO-inducible shRNA targeting ATF4 or scrambled control. ATF4 transcript variant 2, but not variant 1, was present in both cell lines. Knockdown after 96 h of doxycycline treatment was confirmed by PCR. Quantitation of PCR band intensity from three independent experiments expressed relative to SCR control for each cell line. (E) After 96 h of pre-treatment with doxycycline to establish ATF4 knockdown, cells were exposed to reovirus and GSK2606414 for 48 h before analysis of reovirus μ1C protein levels by western blot. (F) Cell were treated with DMSO control, GSK2606414, or thapsigargin in combination with reovirus and analyzed at 48 h for μ1C protein levels by western blot. Data are ±SEM of a minimum of three independent experiments with statistical analysis by unpaired t test, *p < 0.05.