(A) Duplicate NM shRNA and SNAP23 shRNA 3T3L1 adipocytes 8 days after differentiation were immunoblotted for BAX, BAK, and actin. The actin immunoblot is from the same samples run on parallel gels. Immunoblots shown are representative of 4 independently performed experiments. (B) BAX mRNA levels were quantified by qRT-PCR from 4 independent determinations. Data represent the mean ± SEM. (C) Control NM shRNA differentiated adipocytes were treated with 5 μM MG132 or 20 nM bafilomycin A1 for the indicated durations. Cell extracts were prepared and immunoblotted for BAX protein and actin. Immunoblots are representative of 6 independently performed experiments. (D) NM shRNA and SNAP23 shRNA cells under NR conditions were treated with 20 μg/ml CHX and at the time points indicated were immunoblotted for BAX and actin. The NM shRNA BAX immunoblot is from the same samples run on parallel gels. Immunoblot is representative of 3 independent experiments. (E) In the context of the SNAP23 shRNA, the NIH3T3 cells were transfected with nonspecific siRNA or BAX-specific siRNA. Cells were then subjected to ND conditions for 2 hours in the presence and absence of lysosomotropic agents and immunoblotted for the indicated proteins. A relatively lighter and darker exposure for the LC3 immunoblot is shown. Immunoblot is representative of 3 independent experiments. (F) Nonspecific siRNA and BAX siRNA–knockdown cells in the context of the control NM shRNA and SNAP23 shRNA cells were subjected to ND conditions for 6 hours. The cells were then labeled for PI and DAPI. Scale bars: 200 μm. (G) Quantification of PI-positive nuclei was determined by counting 500 cells from 3 independent determinations per genotype. Data represent the mean ± SEM. **P < 0.01, by ANOVA with Tukey’s post hoc test.