Volume 37, Issue 4, Pages 337-349 (May 2016) The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis  Megan L. Goodall, Brent E. Fitzwalter, Shadi Zahedi, Min Wu, Diego Rodriguez, Jean M. Mulcahy-Levy, Douglas R. Green, Michael Morgan, Scott D. Cramer, Andrew Thorburn  Developmental Cell  Volume 37, Issue 4, Pages 337-349 (May 2016) DOI: 10.1016/j.devcel.2016.04.018 Copyright © 2016 Elsevier Inc. Terms and Conditions

Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 MAP3K7 Loss Correlates with TRAIL Receptor Loss, but Not Other Death Receptor Loss, in Prostate Cancer (A) Cell viability of MAP3K7 floxed (white bars) and null (black bars) MPECs treated with chemotherapeutics for 24 hr. Data represent mean ± SD. N.S., not significant. (B) Frequency of homozygous deletions of MAP3K7, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, and FAS in three prostate cancer datasets. (C) Co-occurrence and mutual exclusivity between MAP3K7, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, and FAS. (D) Oncoprint of MAP3K7, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, and FAS (TCGA, n = 333). Each bar represents a patient sample with 150 samples; the additional 183 had no changes to these genes. See also Figure S1. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Necroptotic Cell Death Is Ripk1 Dependent (A) Cell viability of Map3k7 floxed (white bars) and null (black bars) MPECs treated with TRAIL, QVD, and necrostatin-1 for 24 hr. Data represent mean ± SD. (B) Map3k7 null MPECs treated with TRAIL, staurosporine, QVD, and necrostatin-1 and imaged in the presence of annexin V and PI on an IncuCyte in four fields of view every 4 hr for 24 hr, with representative images at 4 hr and scale bars representing 30 μm. Co-localization of annexin V and PI was determined using CellProfiler and the Pearson correlation coefficient (r) was determined. See also Figure S2. (C) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 for 24 hr with Ripk1 shRNA (black bars). Data represent mean ± SD. (D) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, GSK′872, and necrostatin-1 for 24 hr. Data represent mean ± SD. (E) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 for 24 hr with Ripk3 shRNA (black bars). Data represent mean ± SD. (F) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 for 24 hr with Mlkl shRNA (black bars). Data represent mean ± SD. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Autophagy Machinery Localizes with Necrosome Proteins (A) Cell viability of Map3k7 null MPECs treated with TRAIL, necrostatin-1, CQ, BafA1, or wortmannin for 24 hr or with Atg5 and Atg7 shRNA. Immunoblots show knockdown of Atg5 and Atg7 with β-actin as loading control. Data represent mean ± SD. (B) Immunoblots of necrosome proteins and autophagy proteins, ATG7, ATG5/12, p62, BECLIN-1, ULK1, STX17, and LAMP2, after a co-immunoprecipitation of RIPK1 in Map3k7 null MPECs with and without TRAIL treatment. (C) Proximity ligation assay (PLA) of p62 with MLKL and phospho-MLKL in Map3k7 null MPECs treated with and without TRAIL. Scale bars represent 20 μm. (D) Quantification of (C). Data represent mean ± SD. (E) Dual PLA of ATG5 with RIPK1 (red) and ATG5 with MLKL (green) in Map3k7 null MPECs treated with TRAIL or necrostatin. Scale bars represent 20 μm. (F) Quantification of (D). Data represent mean ± SD. (G) Immunogold TEM in Map3k7 null MPECs treated with and without TRAIL. Large gold particles indicated by black arrows recognize antibodies to RIPK1, and small gold particles indicated by white arrows recognize antibodies to ATG5. Scale bars represent 100 nm. (H) Quantification of (G). Data represent mean ± SD. See also Figure S3. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Atg5 Is Required for Necrosome Activation (A) Immunoblots of ATG5, p62, and MLKL after a co-immunoprecipitation of RIPK1 in Map3k7 null MPECs with knockdown of Atg5, with and without TRAIL treatment. (B) PLA of p62 with MLKL and pMLKL in Map3k7 null MPECs treated with and without TRAIL with knockdown of Atg5. Scale bars represent 20 μm. See also Figure S4. (C) Quantification of (B). Data represent mean ± SD. N.S., not significant. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 p62 Is Required for Ripk1 Association with Autophagosome (A) Immunoblots of ATG5, p62, and MLKL after a co-immunoprecipitation of RIPK1 in Map3k7 null MPECs with knockdown of p62/Sqstm1, with and without TRAIL treatment. (B) Dual PLA of ATG5 with RIPK1 (red) and ATG5 with MLKL (green) in Map3k7 null MPECs with knockdown of p62 treated with and without TRAIL. Scale bars represent 20 μm. (C) Quantification of (B). Data represent mean ± SD. (D) Immunogold TEM in Map3k7 null MPECs with p62/Sqstm1 shRNA treated with and without TRAIL. Large gold particles indicated by black arrows recognize antibodies to RIPK1, and small gold particles indicated by white arrows recognize antibodies to ATG5. Scale bars represent 100 nm. (E) Quantification of (D). Data represent mean ± SD. N.S., not significant. See also Figure S5. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 p62 Regulates the Switch between Apoptotic and Necroptotic Cell Death (A) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 with knockdown of p62/Sqstm1 (white bars). Data represent mean ± SD. (B) Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 with p62/Sqstm1 knockdown and imaged in the presence of annexin V and PI on an IncuCyte in four fields of view every 4 hr for 24 hr. Representative images at 4-hr time point with scale bars representing 100 μm. Co-localization of annexin V and PI was determined using CellProfiler and the Pearson correlation coefficient (r) was determined. See also Figure S6. (C) Cell viability of Map3k7 null MPECs treated with TRAIL, QVD, and necrostatin-1 with knockdown of p62/Sqstm1 and addition of either wild-type (WT) p62, mutant p62 lacking the ZZ domain (ΔZZ), or mutant p62 of the LIR domain (ΔLIR). Data represent mean ± SD. N.S., not significant. Developmental Cell 2016 37, 337-349DOI: (10.1016/j.devcel.2016.04.018) Copyright © 2016 Elsevier Inc. Terms and Conditions