Skip to main content
. Author manuscript; available in PMC: 2014 Jun 17.
Published in final edited form as: Cancer Cell. 2013 Mar 28;23(4):435–449. doi: 10.1016/j.ccr.2013.02.017

Figure 6. BCLAF1 promotes autophagy.

Figure 6

(A) Immunoblot analysis of LC3, BCLAF1, and β-actin in SKMM1 myeloma cells induced to express BCLAF1-DHFR for the indicated times. The autophagy-associated LC3-II isoform is indicated. (B) Electron micrographs of UTMC2 myeloma cells induced to express BCLAF1-DHFR for 3 days, or transduced with empty vector. Higher-power images (bottom) corresponding to boxed areas show autophagosomes with double membrane structure (arrows) and autophagolysosomes with degraded organelles (arrowheads). Scale bars: 1 μm. (C) Representative confocal images of SKMM1 myeloma cells expressing mCherry-GFP-LC3B and induced for BCLAF1-DHFR expression for the indicated times. Scale bar: 10 μm. (D) Two myeloma lines (SKMM1, KMS12) were first infected with retroviruses expressing BCLAF1-DHFR or with an empty vector, superinfected with retroviruses expressing an ATG5, ATG7 or control shRNAs and induced for shRNA expression for 2 days prior to BCLAF1-DHFR induction for the indicated periods of time. Live calcein+, PI− cells were quantified by FACS and data were normalized to DMSO values. Shown are means ± SEM. *p<0.05. Data are representative of 3 independent experiments. (E) KMS12 myeloma cells were treated with Q-VD-OPH (25 μM) or with vehicle for 48 hr, and then BCL2 immunoprecipitates were analyzed by immunoblotting for beclin-1 and BCL2. Mouse IgG was used as an immunoprecipitation control. Input proteins were also analyzed. (F) SKMM1 myeloma cells were induced to express shBCLAF1-1 for 3 days prior to Q-VD-OPH (25 μM) or vehicle treatment for 36 hr, and then BCL2 immunoprecipitates were analyzed by immunoblotting for BCLAF1 and BCL2. (G) SKMM1 myeloma cells were transduced with BCLAF1-DHFR or with an empty vector and induced to express BCLAF1-DHFR for 48 hr. Immunoprecipitates prepared using anti-BCL2 antibody-conjugated agarose beads were analysed by immunoblotting for BCLAF1, Beclin-1 or BCL2. A control precipitate prepared from SKMM1 cells transduced with an empty vector was also analyzed as were lysates. See also Figure S6.