ROS-Mediated 15-Hydroxyprostaglandin Dehydrogenase Degradation via Cysteine Oxidation Promotes NAD+-Mediated Epithelial-Mesenchymal Transition  Weixuan Wang, Yadong Hu, Xiaofei Wang, Qingtao Wang, Haiteng Deng  Cell Chemical Biology  Volume 25, Issue 3, Pages 255-261.e4 (March 2018) DOI: 10.1016/j.chembiol.2017.12.008 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2018 25, 255-261. e4DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Decreased NAD Promotes Epithelial-Mesenchymal Transition (A and B) Western blot analysis confirmed CD38 overexpression (CD38(+)) in A549 (A) and SW480 (B) cells. (C) Cellular NAD and NADH levels in A549 CD38(−) and CD38(+) cells. (D and E) Morphological changes of CD38(−) (D) and CD38(+) A549 (E) cells. Scale bar, 50 μm. (F) Western blotting of ZO-1, vimentin, β-catenin, E-cadherin, N-cadherin, cytokeratin 18, and actin expression in CD38(−) and CD38(+) A549 cells. (G) Invasion capability of CD38(−), CD38(+), untreated, and FK866-treated A549 cells as determined by cell invasion assay. (H) Western blotting of ZO-1, β-catenin, E-cadherin, N-cadherin, cytokeratin 18, and actin expression in untreated and 500 μM nicotinic acid-treated CD38(+) A549 cells. Data were analyzed using Student's t test whereby p < 0.05 is considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001; n = 3. All values represent mean from three biological replicates ± SEM. See also Figure S1. Cell Chemical Biology 2018 25, 255-261.e4DOI: (10.1016/j.chembiol.2017.12.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Decreased NAD Levels Increased Cellular Prostaglandin E2 and Promoted 15-PGDH Degradation (A) Prostaglandin E2 (PGE2) levels in CD38(−) and CD38(+) A549 cells (n = 4). (B) Western blotting of 15-hydroxyprostaglandin dehydrogenase (PGDH) expression in CD38(−) and CD38(+) populations of A549 and SW480 cells. (C) Western blotting of 15-PGDH expression in untreated and FK866-treated cells. (D) Western blotting of 15-PGDH expression in untreated, and 500 μM nicotinic acid-treated CD38(+) A549 cells. (E) Cellular NAD and NADH levels in untreated, nicotinic acid-treated, and nicotinamide-treated CD38(+) A549 cells (n = 3). (F) Western blotting of time-dependent decrease in 15-PGDH after A549 cells were treated with 100 nM FK866. Data were analyzed using Student's t test whereby p < 0.05 is considered statistically significant. ***p < 0.001. All values represent mean from at least three biological replicates ± SEM. See also Figure S2. Cell Chemical Biology 2018 25, 255-261.e4DOI: (10.1016/j.chembiol.2017.12.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Reactive Oxygen Species Triggered 15-PGDH Degradation (A) Cellular reactive oxygen species (ROS) levels in CD38(−) and CD38(+) A549 cells. (B–D) Western blotting of 15-PGDH expression in untreated and H2O2-treated A549 cells with or without prior or co-treatment with (B) 6 mM NAc, (C) 10 μM MG132, or (D) 100 nM Baf-A1. (E) Tandem mass spectrometry spectrum of the sulfated peptide for identification of Cys44 modifications in 15-PGDH, with inset showing ion intensity of the peptide in the presence or absence of H2O2. (F) Western blotting of FLAG-tagged 15-PGDH, 15-PGDH C44A, 15-PGDH C44D, and endogenous 15-PGDH from untreated and H2O2-treated 15-PGDH(+) A549 cells with 5 s of exposure time. Numbers represent quantitation of western blot image by grayscale analysis. Data were analyzed using Student's t test whereby p < 0.05 is considered statistically significant. ***p < 0.001; n = 3. All values represent mean from three biological replicates ± SEM. See also Figure S2. Cell Chemical Biology 2018 25, 255-261.e4DOI: (10.1016/j.chembiol.2017.12.008) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Degradation of 15-PGDH Promoted EMT (A) mRNA and protein expression levels of 15-PGDH in A549 NCi and PGDH-knockdown cells. (B) Invasion capacity of A549 15-PGDH-knockdown and NCi cells as determined by cell invasion assay. (C) Western blotting of vimentin, ZO-1, β-catenin, cytokeratin 18, 15-PGDH, and actin expression in 15-PGDH-overexpressing and control CD38(+) A549 cells. (D) Invasion capacity of 15-PGDH-overexpressing and control CD38(+) A549 cells as determined by cell invasion assay. (E) Schematic of proposed relationship between aging and tumorigenesis. Data were analyzed using Student's t test whereby p < 0.05 is considered statistically significant. **p < 0.01, ***p < 0.001; n = 3. All values represent mean from three biological replicates ± SEM. See also Figure S3 and Table S1. Cell Chemical Biology 2018 25, 255-261.e4DOI: (10.1016/j.chembiol.2017.12.008) Copyright © 2017 Elsevier Ltd Terms and Conditions