DENV induces rearrangement of the ER early after infection.

<p>2fTGH cells were (A) mock-infected or (B–E) infected with DENV-2 for 12 h and stained intracellularly for UPR markers and viral proteins E and NS3. (A) Mock-infected 2fTGH cells were stained with mouse MAbs against DENV E (4G2; green) and NS3 (E1D8; red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Intracellular staining of DENV-infected cells was carried out as in (A); in addition primary antibodies specific for cellular proteins (B) GRP78, (C) GRP94, (D) XBP1 and (E) ATF6 (magenta) were used, followed by secondary antibodies conjugated to Alexa Fluor® 647. The multiple-exposure images were captured using 63X/1.25 Plan-Neofluar DIC oil objective on Zeiss 510 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647. For individual images, the additional channels were turned OFF post-acquisition using the Zeiss operating software. Co-localization is observed in the “Merged” image as a yellow hue. Merged and DIC images represent a single image with all detector channels ON. Total magnification 630×. (F) Western blot analysis of GRP78 over a 12-h course of DENV infection. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection.