DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection.

<p>2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038202#pone-0038202-g001" target="_blank">Figure 1</a>. (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.