Figure 4

Release of Gaussia luciferase does not require autophagy. A, 293 T cells were treated with 10 μM of digitoxin (Dig), chloroquine (CQ), Trifluorperazine (TFP), rapamycin (Rap), tamoxifen (Tam), or theonyltrifluoracetone (TTFA), 10 μg/mL Brefeldin A, DMSO or left untreated (NT) and analyzed for luciferase activity in supernatants (*P < 0.05; **P < 0.01; ***P < 0.001). B, embryonic fibroblasts from ATG5 inducible knockout mice were transduced with dNGLUC. ATG5 knockout was induced using 1 μg/mL of doxycycline. Cells and supernatants were collected on three consecutive days after induction. Cells were washed and incubated in fresh medium for an additional 2 h before collection of supernatant. The efficiency of ATG5 knockout was verified by immunoblotting with detetction of LC3 (B), β-actin, and ATG5 (C). D, Luciferase activity in the correspondent supernatants was measured using the Perkin Elmer Envision II at the indicated time points. A, B, shown is the mean plus standard deviation of three experiments. Significances were calculated with a two-sided paired t-test. R.L.U. – relative light units, SN - supernatant.