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. 2013 Jun 7;32(16):2204–2216. doi: 10.1038/emboj.2013.133

Figure 4.

Figure 4

XIAP regulates serum starvation-induced autophagy. (A) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time. GFP-LC3 puncta formation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments. (B) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6B. (C) HCT116 XIAP WT and XIAP KO cells were treated with 1 μM API-2 for 6 h. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6C. (D) HCT116 cells were treated with or without EBSS for 4 h. Cell lysates were subjected to immunoprecipitation with anti-XIAP antibody followed by western blot analysis. The data are representative of two biological replicates. (E) Lysates from HCT116 cells were immunoprecipitated with anti-Mdm2 antibody. Immunoprecipitates and cell lysates were analysed by western blotting with anti-p-XIAP antibody. The data are representative of two biological replicates. (F) HCT116 XIAP KO cells were transfected with Flag-XIAP, Flag-XIAP S87A, Flag-XIAP S87D or control vector. Twenty-four hours after transfection, cells were harvested. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6D. (G) p53−/−Mdm2−/− MEF cells were transfected with HA-Mdm2 plus Flag-XIAP, Flag-XIAP S87A or Flag-XIAP S87D. Twenty-four hours later, cells were treated with 20 μM MG-132 for another 4 h. Cell lysates were subjected to immunoprecipitation with anti-HA antibody. Immunoprecipitates were analysed by western blotting. The data are representative of two biological replicates.