## 4 Discussion  
In this study, we found that 4 HR increased TGF-$1 expression through upregulation of stress responses in both mitochondria and the ER in HUVECs. ER stress-associated markers such as GRP78, ATF4, ATF6\(\alpha\), p-eIF2, and p-PERK were increased after 4 HR administration (Figure 2a). The administration of TUDCA, an ER stress inhibitor, attenuated 4 HR-induced TGF-$1 expression (Figure 2c). The mitochondrial-stress-inducing chemical carbonyl cyanide m-chlorophenylhydrazone increases TGF-$1 expression [29]. Figure 5: 4 HR administration and SIRT expression. **(a)** The administration of 4 HR did not change the expression of SIRT1 and SIRT3 in HUVECs. **(b)** The administration of 4 HR increased the expression of SIRT6 in HUVECs. **(c)** Quantitative RT-PCR results showed that the _SIRTS_ mRNA expression level was significantly increased by 4 HR administration (* \(p<0.05\), **\(p<0.01\)** compared to untreated control). **(d)** The administration of 4 HR (1–100 uM) increased pan-SIRTs enzyme activity (* \(p<0.05\) compared to the control group). **(e)** The administration of 4 HR increased NAD+ production (* \(p<0.05\) compared to the control group). **(f)** 4 HR also increased the NAD+/NADH ratio (* \(p<0.05\) compared to the control group). All blots were performed in triplicate, and the statistical analysis of the relative expression level is shown in Supplementary Figure S8. Decreased mitochondrial activity following 4 HR administration reduced both ATP production (Figure 1a) and oxygen consumption (Supplementary Figure S1). As noted in our previous study, 4 HR is a class I HDACi [22]. HDACis induce TGF-\(\beta\)1 expression via activation of Sp1 [20]. In this study, 4 HR increased the expression level of p-Sp1. Hyper-acetylated H3 is also required for the expression of TGF-\(\beta\)1, and this response is enhanced by HDACi [23]. In this study, H3 was hyper-acetylated by 4 HR administration (Figure 3d). 4 HR exhibited a wide variety of functions, such as anti-cancer effects [21], pro-angiogenic effects [8; 17], anti-inflammatory effects [30], and increased bone formation [31]. These divergent effects are likely to be involved in the broad-spectrum regulation of cellular events [24; 32]. In this study, 4 HR increased TGF-\(\beta\)1 expression via hyper-acetylated H3 and p-Sp1 (Figure 5). Sp1 is a transcription factor for TGF-\(\beta\)1, and its expression is increased by histone deacetylase inhibitors [20]. Interestingly, 4 HR also increases Sp1 expression in oral cancer cells and SCC-9 cells [21]. Therefore, increased TGF-\(\beta\)1 expression as a result of 4 HR administration might be associated with its HDACi activity and the subsequent promotion of Sp1 expression (Figure 3). In fact, 4 HR is a class I HDACi [22]. Hyper-acetylated H3 is also required for the expression of TGF-\(\beta\)1, and this response is enhanced by HDACi [23]. Therefore, 4 HR administration might increase acetylated H3. In this study, the Sp1/Sp3 ratio gradually increased at 8, 16, and 24 h after 4 HR administration (Figure 4). In addition, 4 HR administration increased p-Sp1 and TGF-\(\beta\)1 expression (Figure 3a). The administration of thithramycin inhibited 4 HR-induced p-Sp1 and TGF-\(\beta\)1 expression (Figure 3b). Sp1 transcription activity and (Figure 3c) acetylated H3 were also increased by 4 HR administration (Figure 3d). When cells face ER stress, the mechanism for restoring ER homeostasis is activated [8]. As active protein synthesis increases ER loading, protein translation activity is decreased [33]. To neutralize ER stress, the expression of chaperones is increased [34]. Organic chemicals which may induce ER stress can bind to intracellular decoy proteins and change the protein conformation. This process may increase the production of misfolded proteins. ATF6 is activated by ER stress [35], which leads to the removal of misfolded proteins [36]. In this study, the expression level of ATF6\(\alpha\) was increased by 4 HR administration (Figure 2a).