Fig 2: AROS abrogates DNA damage-induced senescence by stabilizing SIRT1.a–c Effects of AROS overexpression on doxorubicin-induced senescence (a), SIRT1 degradation (b), and SIRT1 ubiquitination (c). Doxorubicin was applied to two subclones of A549 cells (F-AROS1 and F-AROS2) that stably express Flag-AROS. a After 4 days, the cells were examined via SA-β-gal staining. The error bars represent the means ± SDs of three independent experiments (**P < 0.01). b Under the same conditions, the SIRT1 levels were monitored via WB using the indicated antibodies. c After the addition of MG-132, SIRT1 ubiquitination was measured via IP using an anti-SIRT1 antibody and WB using an anti-poly-Ub antibody. d–f Effects of AROS knockdown on doxorubicin-induced senescence (d), SIRT1 degradation (e), and SIRT1 ubiquitination (f). AROS-depleted A549 cells were selected using two types of sh expression vectors. Two subclones of AROS-depleted A549 cells were treated with (+) or without (−) doxorubicin. d The numbers of SA-β-gal-positive cells were counted. The error bars represent the means ± SDs of three independent experiments (**P < 0.01). e SIRT1 degradation was monitored via WB using the indicated antibodies. f SIRT1 ubiquitination was monitored as described above. g, h Effects of ectopic AROS overexpression (g) and knockdown (h) on SIRT1 stabilization. HEK293 cells were transiently transfected with Flag-SIRT1, Myc-Cdh1, and Flag-AROS (for overexpression) or sh-AROS (for knockdown). Cell lysates were subjected to WB using the antibodies shown on the right. i Effect of AROS overexpression on Cdh1-promoted SIRT1 ubiquitination. HEK293 cells were transfected as indicated in the presence of MG-132. Ubiquitinated SIRT1 was visualized via IP using an anti-Flag antibody and WB using an anti-HA antibody. M, mock; FE1, Flag-empty #1; FA1, Flag-AROS #1; FA2, Flag-AROS #2; sL1, sh-Luc #1; sL2, sh-Luc #2; sA1-1, sh-AROS #1-1; sA1-2, sh-AROS #1-2; sA2-1, sh-AROS #2-1; and sA2-2, sh-AROS #2-2.

Fig 3: The reciprocal expression patterns of AROS and Cdh1 are associated with pinosylvin-mediated SIRT1 stabilization in bleomycin-induced pulmonary senescence.a Inhibition of bleomycin-induced cellular senescence by pinosylvin. A549 or TIG-3 cells were treated with 25 μM or 50 μM pinosylvin prior to exposure to bleomycin. A549 and TIG-3 cells were treated with bleomycin at concentrations of 5 μM and 2.5 μM, respectively, for 24 h. Bleomycin-induced senescence was monitored via microscopy and quantified by counting the number of SA-β-gal-positive cells. The error bars represent the means ± SDs of three independent experiments (*P < 0.05, **P < 0.01). b Effect of pinosylvin on the p53/p21 senescence pathway in bleomycin-induced senescent cells. The protein expression patterns were examined via WB using the antibodies shown on the right. c Protection against bleomycin-induced mouse pulmonary senescence by pinosylvin in vivo. Mice were treated as described in “Materials and methods”. The level of SA-β-gal staining was analyzed using ImageJ software. Scale bar, 20 μm. d Effect of pinosylvin on the histological and morphometric patterns. Hematoxylin and eosin (H&E) staining and morphometric analysis of the alveolar space in PBS (n = 5), Pino (n = 5), BM (n = 9), and BM/Pino (n = 8)-administered lung sections. e Masson’s trichrome staining of PBS-, Pino-, BM-, and BM/Pino-administered lung sections. The error bars show the means ± S.E.Ms.; ns, not significant; ***P < 0.001 and ****P < 0.0001, two-tailed Student’s t-test. Scale bars: 200 μm (d) and 100 μm (e). f Effect of pinosylvin on the expression patterns of genes associated with the p53/p21 and p16 pathways. The protein expression patterns were determined via WB using the indicated antibodies. g Effect of pinosylvin on the expression patterns of AROS and Cdh1. After combined treatment with pinosylvin and bleomycin, mouse lung tissues were subjected to IHC staining using antibodies against SIRT1, AROS, and Cdh1. PBS (n = 4–5), Pino, BM, and BM/Pino (n = 5). The stained images were analyzed using ImageJ software. Scale bar, 20 μm. The error bars represent the means ± SDs of experiments using four mice (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: AROS competes with Cdh1 to bind and stabilize SIRT1.a Schematic diagram of competition between AROS and Cdh1 for SIRT1 binding. Amino acids 114–217 in SIRT1 comprise the AROS-binding site. SIRT1∆ represents the deletion of amino acids 1–217 of SIRT1. b Requirement of amino acids 1–217 of SIRT1 for Cdh1 binding. HEK293 cells were transfected with Myc-Cdh1 and Flag-SIRT1 WT or Flag-SIRT1∆. Cell lysates were subjected to IP using an anti-Myc antibody and WB using an anti-Flag antibody. c Direct interaction of the SIRT1 fragment with AROS and Cdh1. Purified GST, GST-AROS, or GST-Cdh1 protein was incubated with in vitro-translated Flag-SIRT1∆ or the Flag-SIRT1(114–217) fragment. The bound proteins were visualized via WB using an anti-Flag antibody. d Requirement of amino acids 1–217 of SIRT1 for Cdh1-mediated SIRT1 turnover. HCT116 cells were transiently transfected with Myc-Cdh1 and GFP-SIRT1-WT or GFP-SIRT1∆ and further treated with CHX (50 μg/ml) for the indicated time periods. Images were visualized by WB using individual antibodies. The error bars represent the means ± SDs of three independent experiments (*P < 0.05). e Competition for SIRT1 binding. GST or GST-SIRT1(114–217) and in vitro-translated Myc-Cdh1 were incubated with increasing amounts of in vitro-translated Flag-AROS. The bound proteins were visualized via WB using the indicated antibodies. f Competition for SIRT1 stability. HEK293 cells were transfected with Flag-SIRT1, Myc-Cdh1, and increasing amounts of Flag-AROS. The protein levels were monitored by WB using the indicated antibodies.

Fig 5: Schematic representation of the roles of AROS and pinosylvin in Cdh1-mediated SIRT1 ubiquitination during stress-induced premature senescence.DNA damage signaling triggers the activation of Cdh1, which leads to SIRT1 degradation and subsequent induction of premature senescence. AROS abrogates SIPS by suppressing Cdh1-mediated SIRT1 ubiquitination and thereby activates SIRT1; these effects cause p53 inactivation and the downregulation of SASP-associated genes. Pinosylvin has a protective effect against bleomycin-induced pulmonary senescence through its function as a SIRT1 activator.