Supplementary Information CYLD Negatively Regulates Transforming Growth Factor-β Signaling via Deubiquitinating Akt Jae Hyang Lim, Hirofumi Jono, Kensei Komatsu, Chang-Hoon Woo, Jiyun Lee, Masanori Miyata, Takashi Matsuno, Xiangbin Xu, Yuxian Huang, Wenhong Zhang, Soo Hyun Park, Yu-Il Kim, Yoo-Duk Choi, Huahao Shen, Kyung-Sun Heo, Haodong Xu, Patricia Bourne, Tomoaki Koga, Haidong Xu, Chen Yan, Binghe Wang, Lin-Feng Chen, Xin-Hua Feng and Jian-Dong Li 1
Supplementary Figure S1. CYLD prevents development of lung fibrosis independent of p38 MAPK following S. pneumoniae infection. (a) Relative quantity of mrna expression of connective tissue growth factor (CTGF), type I collagen (COL1A2), and type 1 plasminogen activator inhibitor (PAI-1) compared to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured in the lung tissues of Cyld+/+ and Cyld-/- mice 2 weeks post S. pneumoniae infection (5 106 CFU/mouse). *p<0.05. Values are the means ± s.d. (n = 3). Un-paired Student s t-test was used for comparison with S. pneumoniae in Cyld+/+. (b) Cyld-/- mice were first i.t. inoculated with S. pneumoniae with vehicle control or p38 MAPK inhibitor (SB203580, 20 mg/kg body weight) for 2 weeks, and lung tissues were then collected from mice survived from lung injury and histopathological analysis was performed (n=5 for CON, 5 for SB203580, 20 for S. pneumoniae infection with vehicle control, and 15 for S. pneumoniae with SB203580). Scale bars correspond to 200 µm. (c) Wild-type (WT) mice were inoculated with S. pneumoniae for various times as indicated in the figure. Total- and phospho-p38 MAPK in the lung tissue protein was 2
measured using total and phospho-p38 ELISA kit (Invitrogen), and TGF-β concentration was measured in blood using mouse TGF-β ELISA kit (R&D Systems). *p<0.05 compared to control, **p<0.001 compared to control. Values are the means ± s.d. (n = 5). Un-paired Student s t-test was used for comparison with 0 hour Control. 3
Supplementary Figure S2. Expression of CYLD is lower in fibrotic lung tissue. (a) Lung fibrosis tissues were obtained from the patients with pulmonary fibrosis during pneumonectomy, and control tissues were obtained from the patient with pneumothorax during the surgery. Lung tissue sections were stained with H&E, Masson s trichrome (Trichrome), anti-cyld, and anti-smad3. Slides are representative of 5 (CON) and 10 (Fibrotic Lung) human lung tissues. Scale bars correspond to 200 µm. (b) WT mice were i.t. inoculated with TGF-β (50 ng or 100ng), and relative quantity CYLD mrna expression was measured in the lung tissues of mice. Values are the means ± s.d. (n=3). 4
Supplementary Figure S3. CYLD-deficiency enhances Bleomycin-induced lung fibrosis. Cyld+/+ and Cyld-/- mice were i.t. inoculated with bleomycin (3 Units/kg body weight) for 2 weeks, and lung tissues were collected from mice survived from lung injury and subjected to histopathological analysis (H&E stain and Trichrome stain) (n=5 for CON in Cyld+/+ and Cyld-/- mice, 40 in bleomycin in Cyld+/+ mice, and 35 in bleomycin in Cyld-/- mice). Scale bars correspond to 200 µm. 5
Supplementary Figure S4. sichip reduces CHIP mrna expression. Relative quantity of mrna expression of CHIP compared to GAPDH was measured in A549 cells transfected with sicon or sichip. Values are the means ± s.d. (n = 3). 6
Supplementary Figure S5. CYLD interacts with Akt but not with CHIP or GSK3β. Cells were co-transfected with Flag-CYLD, Myc-CHIP, HA-GSK3β, or Flag-Akt, and CYLD was pulled down with anti-cyld antibody. Interacting proteins were analyzed by immunoblotting with the indicated antibodies. 7
Supplementary Figure S6. Akt mediates CYLD-mediated negative regulation of TGFβ signaling. (a) MEF cell extracts from Cyld +/+ and Cyld -/- mice were analyzed by immunoblotting with the indicated antibodies. (b) CYLD was reduced with sicyld in MEF cells from Akt1 +/+ and Akt1 -/- mice, and relative quantity of mrna expressions of PAI-1 compared to GAPDH was measured following TGF-β treatment. Values are the means ± s.d. (n = 3). 8
Supplementary Figure S7. Akt induces fibrotic responses via Smad3. MEF cells from Smad3 +/+ and Smad3 -/- mice were transfected with C/A-Akt, and relative quantity of PAI- 1 and CTGF mrna expression compared to GAPDH was measured by real-time Q-PCR analysis. Values are the means ± s.d. (n = 3). 9
Supplementary Figure S8. S. pneumoniae induces endogenous Akt ubiquitination, and CYLD deubiquitinates it. MEF cells from Cyld +/+ and Cyld -/- mice were treated with S. pneumoniae, and Akt in cell lysate was pulled down with anti-akt antibody, and immunoblotted against Ub, CYLD, and Akt. 10
Supplementary Figure S9. CYLD inhibits TGF-β signaling independent of TRAF6. TGF-β-induced SBE-Luc activity was measured in TRAF6-depleted using sitraf6 with or without sicyld. Human sirna for TRAF6 was from Dharmacon, and knockdown of TRAF6 using sitraf6 was performed with Lipofectamine 2000 (Invitrogen). ON- TARGETplus SMARTpool of sirna targeting human TRAF6 consists of four sirnas and sequences for the sirnas are as follows: 5 -GGAGACAGGUUUCUUGUGA-3, 5 - GAUAUGAUGUAGAGUUUGA-3, 5 -GGCCAUAGGUUCUGCAAAG-3, 5 - GCGCUUGCACCUUCAGUUA-3. *p<0.001. Values are the means ± s.d. (n = 3). Statistical data analysis was performed using Student s t-test. 11