Fig 1. Bax reduces autophagosome formation.

(a) Bcl-xL (0.6ug), Bcl-xL (0.6ug)/Beclin 1 (0.6ug), Bcl-xL (0.6ug)/Beclin 1 (1.2ug)/Bad (0.6ug) and Bcl-xL (0.6ug)/Beclin 1 (1.8ug)/Bax (0.6ug) were transfected into HeLa cells. To achieve similar expression levels of Bcl-xL and Beclin 1, we used different amounts of Bcl-xL and Beclin 1 in each transfection. Empy vectors were used to keep the amount of DNA in each transfection constant. After 20 hours, cells were lysed and anti-Flag was used for immunoprecipitation. Immunoprecipitates were detected with anti-Bcl-xL and anti-Flag (Beclin 1), respectively. Total lysates were detected with anti-Bcl-xL, anti-Flag, anti-Bad and anti-Bax, respectively.

(b) HeLa cells were transfected with empty vector (lanes 1, 3), or Bax (lanes 2, 4). After 20 hours, one of the two sets (lanes 3-4) was treated with Bafilomycin A1 (Baf) for 4 hours. This treatment blocks LC3-II degradation and is saturating; thus, the changes in LC3-II reflect altered autophagosome synthesis ⁸. The cells were then lysed and subjected to SDS-PAGE and blotting with anti-LC3 (top), anti-tubulin (bottom) respectively. Note that lighter exposures were used in the Baf conditions to enable assessment of changes in LC3-II levels, since Baf dramatically increases LC3-II levels. Endogenous LC3-I levels appear very low in HeLa cells as LC3 antibody has stronger immunoreactivity to LC3-II than LC3 I ³³.

(c) GFP-LC3 was co-transfected with increasing levels of Bax plasmid, as indicated in duplicate. (In all experiments of this type, the total amount of transfected DNA is kept constant by using empty vector.) After transfection, one set were treated with DMSO (lanes 1-6) and the other set were treated with z-VAD-fmk (20uM) (lanes 7-12). After 20 hours, cell lysates were subjected to western blot with anti-GFP (top and middle) and anti-tubulin (bottom).

(d) GFP-LC3/vector, or GFP-LC3/Bax were transfected into HeLa cells. The GFP-LC3/Bax transfections were treated with either DMSO or z-VAD-fmk (20uM), and GFP-LC3 transfection was also treated with DMSO following transfection. After 20 hours, cells were fixed and the numbers of GFP puncta were scored. Pictures were taken under fluorescent microcope. These transfections do not give rise to GFP-LC3 aggregates ⁵. *: P1<0.05; P2<0.01.

(e) Vector or Bax were transfected into HeLa cells. The Bax transfections were treated with either DMSO or caspase-3 inhibitor (Ac-DEVD-CHO) (20uM), and vector transfection was also treated with DMSO following transfection. In a parallel experiment, cells were treated with Bafilomycin A1 for the last 4 hours prior to harvesting. After 20 hours, cell lysates were subjected to western blots and probed with anti-LC3, anti-tubulin antibodies. LC3-II densitometry is indicated (versus tubulin).