Extended Data Fig. 2: cIAP1/2 and TRAF2 are required for induction of autophagy by ICCB-19/Apt-1.
From: Modulating TRADD to restore cellular homeostasis and inhibit apoptosis
a, HEK29T cells were transfected with Flag-Beclin 1 for 12 h, then treated with Apt-1 (10 μM) for another 12 h. Cell lysates were immunoprecipitated using anti-Flag beads. cIAP1 and TRAF2 levels were determined by immunoblotting. b, MEFs were treated with indicated concentrations of Apt-1 for 12 h. Cell lysates were immunoprecipitated using anti-Beclin 1 antibody. cIAP1 and TRAF2 levels were determined by immunoblotting. c, d, Long-lived protein turnover rates in MEFs with indicated genotypes treated with indicated compounds. Expressed as fold changes relative to normal control cells. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. ***P = 3 × 10−14, 6 × 10−10, 1 × 10−7, 2 × 10−8 (left to right, c, d). e, MEFs were pre-treated with SM-164 (1 μM) for 1 h, then treated with Apt-1 for 6 h. LC3 II levels were determined by immunoblotting. Mean ± s.e.m. of n = 3 biologically independent experiments. Two-tailed t-test. ***P = 0.0003. f, shRNA-mediated TRAF2 stable knockdown MEFs were treated with Apt-1 (10 μM) for 6 h. LC3 II levels were determined by immunoblotting. Mean ± s.e.m. of n = 3 biologically independent experiments. Two-tailed t-test. **P = 0.0012. g, h, cIap1−/− and Traf2−/− MEFs reconstituted with HA-mcIAP1 and HA-mTRAF2, respectively, were treated with Apt-1 (10 μM) for 6 h. LC3 II levels were determined by immunoblotting. Mean ± s.e.m. of n = 3 biologically independent experiments. Two-tailed t-test**P = 0.0057 (g), ***P = 0.0007 (h). i, j, MEFs with indicated genotypes were treated with rapamycin (1 μM) for indicated time. LC3 II levels were determined by immunoblotting. The quantification of each experiment was shown on the right (n = 1), representative of 3 independent experiments. k, l, MEFs with indicated genotypes were incubated in HBSS for indicated time. LC3II levels were determined by immunoblotting. The quantification of each experiment was shown on the right (n = 1), representative of 3 independent experiments. m, MEFs were treated with indicated compounds for 6 h, then cell lysates were tandem-immunoprecipitated with anti-Beclin 1 antibody and denatured in 3 M urea. Anti-K63-linkage specific polyubiquitin antibody was used to conduct secondary immunoprecipitation. Samples were then immunoblotted with anti-Beclin 1 antibody to measure the K63-linkage specific ubiquitination of Beclin 1. n, MEFs were pretreated with SM-164 (1 μM) for 1 h, then treated with Apt-1 (10 μM) for 6 h, then K63-linkage specific ubiquitination of Beclin 1 was analysed as in (m). o, p, Reconstituted MEFs were treated with Apt-1 (10 μM) for 6 h, then K63-linkage specific ubiquitination of Beclin 1 was analysed as in (m). For gel source data, see Supplementary Fig. 1.
