BNIP3 induction in HEK 293 cells reduces cell growth.

HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Cells were untreated or treated with doxycycline (1 microg/mL) to induce gene expression. (A) Reactive oxygen species (ROS) production was increased upon BNIP3 (but not β-gal) induction. ROS were detected using the dye CM-H2DCFDA, which is oxidized to green fluorescent DCF (dichlorofluorescein) by hydrogen peroxide. (B) Autophagy was also increased upon BNIP3 (but not beta-gal) induction, as determined by GFP-LC3 localization. TO-cells were transiently transfected with GFP-LC3. 24 hours after transfection, cells were untreated or treated with doxycycline for an additional 24 hours. (C) Cell death was not induced upon BNIP3 or β-gal induction, as determined by the membrane permeability assay. (D) Cell growth was severely restricted upon BNIP3 (but not beta-gal) induction in HEK293 cells. For each cell type, 1.5x105 cells were seeded in one well of a 6-well culture dish, with or without doxycycline (gene induction). Three days later, the number of adherent cells in each well was determined using a Beckman Coulter Counter. The fold-increase represents the ratio of cells on day three compared to the number of cells originally seeded. Results represent the average of three independent experiments; error bars represent standard deviation. *p < .01.