Fig 2: cIAP2 and XIAP regulate autophagosome fusion, but not cIAP1.a MEFs expressing mCherry-GFP-LC3b were transfected with siRNA against either cIAP1, cIAP2 or XIAP. Cells were analysed with the microscope and the number of mCherry+, and GFP + puncta/cell were calculated and the ratio of GFP + /mCherry + puncta is indicated. Shown are the means and the error bars represent the SEM of at least three independent experiments. Westerns show efficient knockdown of cIAP1 and XIAP expression. cIAP2 siRNA efficiency was determined by real time PCR as shown in the graph below the westerns. b Wild type and cIAP2-/- XIAP-/- dermal fibroblasts expressing mCherry-GFP-LC3b were treated analysed on the microscope and mCherry+, and GFP + puncta/cell were calculated. The ratio of GFP+/mCherry + puncta is indicated. c Wild type and cIAP2-/- XIAP-/- dermal fibroblasts were left in complete media (CM) or starved for 2 h in EBSS. Cells were lysed and proteins analysed by western blot for XIAP, LC3, and Actin. cIAP2-/- was confirmed by PCR due to lack of effective antibodies for mouse cIAP2 (see supplemental Fig. 2). d Immunofluoresence showing accumulation of LC3 in starved cIAP2-/- XIAP-/- dermal fibroblasts. Wild type and cIAP2-/- XIAP-/- dermal fibroblasts were incubated in complete media or starved in EBSS for 2 h, then fixed and stained for LC3 (green channel) and LAMP2 (red channel). Nuclei are stained blue with Hoechst. Shown in upper panels are overviews. Lower panels show zoomed in regions indicated in the upper panels

Fig 3: Effect of VP-16 on the induction of apoptosis in LX-2 cells.(a) LX-2 cells were stained with JC-1 after incubation with VP-16 for 72 h to determine ??m (magnification × 400). Undamaged mitochondria are hyperpolarized and stained red by the JC-1 dye. When mitochondria are damaged by VP-16, ??m decreases, and the JC-1 dye diffuses out of the mitochondria. Thus, the dye shows green fluorescence. (b) LX-2 cells were treated with various concentrations of VP-16 for 72 h. Apoptotic cells were analyzed using flow cytometry. The percentage of Annexin V-positive cells relative to untreated controls is indicated in a bar chart. (c) LX-2 cells were treated as described above. Western blotting was used to analyze the expression of PARP, cleaved PARP, XIAP, caspase-9, caspase-7, cytochrome c, caspase-3 and cleaved caspase-3. ß-actin was used as a loading control. VP-16 increased the caspase-3 activities of LX-2 cells, which was assessed using a Caspase-3 Activity Assay kit. (d,e) LX-2 cells were pretreated with z-VAD-FMK (40 µM) for 1 h and then with 4 µM VP-16 for 72 h. (d) Annexin V-positive cells were examined by flow cytometry. PARP and cleaved PARP were measured by western blotting. ß-actin was used as the loading control. (e) Cell viability was assessed using the CCK-8 assay. The results are expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.