Nf κb

For validation of primary hits from the high-throughput screen, cDNA clones were expanded from bacterial stocks of the library, and DNA was isolated using a HiSpeed Maxi Kit (Qiagen). Each clone was sequence verified and retested in six replicate wells in 384-well format using the conditions described above for the full screen. Each clone was also tested for its ability to regulate STAT1 in uninfected cells, as well as its ability to trigger a control luciferase reporter lacking GAS elements. Additional reporters were also used for monitoring ISGF3 (Stratagene), IRF1 (ActiveMotif), NF-κB, SRF, and AP1 (Clontech), as described in the text.

Briefly, cells (2.5 × 10⁵ per well) were seeded in 6-well plates and transfected overnight with 1 μg of NFκB, TOPLASH, or FOPFLASH reporter construct (Clontech, Palo Alto, CA) and cotransfected with 0.5 μg of β-galactosidase expression plasmid (pSV-β-gal; Promega, Madison, WI), using Lipofectamine 2000 (Life Technologies). 24 h after transfection, cells were treated with exosomes with or without HECD-1 blocking antibodies for 6 h, and then harvested with reporter lysis buffer (Promega). Luciferase activity was assayed using the luciferase assay kit according to the manufacturer’s protocol (Promega). β-galactosidase activity was used to normalize the transfection.

To evaluate the promoter activity regulated by EcAtg5, luciferase reporter plasmids, including interferon-sensitive response element (ISRE)-Luc, IFN3-Luc, and nuclear factor (NF)-κB (Clontech, USA), were used for co-transfection. Briefly, GS cells were transiently transfected with the luciferase plasmids together with the indicated EcAtg5 expression vectors using Lipofectamine 2000 reagent. The pRL-SV40 Renilla luciferase vector was used as an internal control. Luciferase activity of total cell lysates was measured using a luciferase reporter assay system (Promega).