Extended Data Fig. 6: Autophagy is required for ICCB-19/Apt-1 to inhibit Velcade-induced apoptosis.
From: Modulating TRADD to restore cellular homeostasis and inhibit apoptosis
a, Tradd+/+ and Tradd−/− MEFs were treated with vehicle or ICCB-19 (10 μM) for 6 h. Autophagy levels were determined by immunoblotting using anti-LC3 antibody. Mean ± s.e.m. of n = 3 biologically independent experiments (right). Two-tailed t-test. n.s. not significant, (P = 0.9172). b, Tradd+/+ and Tradd−/− MEFs were treated with Apt-1 (10 μM), NH4Cl (10 mM) as indicated for 6 h. Autophagy levels were determined by immunoblotting using anti-LC3 antibody. Mean ± s.e.m. of n = 3 biologically independent experiments. Two-tailed t-test. n.s. not significant (P = 0.4064, 0.8913 (left to right)). c, Long-lived protein turnover rates in Tradd+/+ and Tradd−/− MEFs. Expressed as as fold changes relative to Tradd+/+ cells. Mean ± s.e.m. of n = 5 biologically independent samples, representative of 3 independent experiments. Two-tailed t-test. ***P = 0.0001. d, WT and Tradd-KO Jurkat cells were treated with Apt-1 (10 μM) and Spautin-1 (10 μM) followed by Velcade (50 nM) for 24 h. Cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. ***P = 2 × 10−5, 3 × 10−15 (left to right). e, Jurkat cells were treated with ICCB-19 (10 μM), Apt-1 (10 μM), Chloroquine (50 μM), E64d (5 μg/ml) followed by Velcade (50 nM) for 24 h. The cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. ***P = 4 × 10−14, 1 × 10−15 (left to right). f, Atg5-WT and Atg5-KO Jurkat cells were pretreated with Apt-1 (10 μM) or zVAD (20 μM) for 1 h, then stimulated by Velcade (50 μM) for 24 h. Cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. ***P = 1 × 10−15. Validation of Atg5 knockout was determined by immunoblotting and shown on the right. g, Atg5+/+ and Atg5−/− MEFs were stimulated by TNF (1 ng/ml) and 5z7 (0.5 μM) for 8 h in the presence or absence of Apt-1 (10 μM). Cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. n.s. not significant, (P = 0.2568, 0.0822 (left to right)). h, HEK293T cells were transfected with indicated expression plasmids for 24 h. The whole-cell lysate lysed in 6 M urea was subjected to pull-down with Ni2+ beads and analysed by immunoblotting with anti-Beclin 1 antibody to detect ubiquitylated Beclin 1. The ubiquitination of Beclin 1 by cIAP1 was reduced upon overexpression of TRADD, which was restored by Apt-1. For gel source data, see Supplementary Fig. 1.
