1 μg/mL demecolcine, a potent mitotic poison, in combination with 100 μM 5HT. After 5 days 5HT in SFM caused 7- to 8-fold higher values of MTT activity in Huh7 and HepG2, whereas Autophagy Compound Library cost serum deprivation led to complete

cell death (Fig. 1D,E). This clearly could not only be attributed to increased cell viability but also to an enhanced rate of proliferation. Initial proliferation in SFM was prevented by demecolcine. The combination of 5HT and demecolcine abolished cell proliferation, but importantly, MTT activity did not drop below the baseline of 100% MTT activity. Thus, we concluded that 5HT predominantly promotes cell survival of Huh7 and HepG2 and this prerequisite facilitates cell proliferation. To identify a potential target receptor that is involved in 5HT-mediated survival of HCC cells we tested different agonists and antagonists. A scheme of the experimental setup is shown in Supporting Fig. 2A. 5HT2 receptors belong to the Gq/11 family

of G-proteins. Their stimulation results in the activation of phospholipase C (PLC) and further downstream in the activation of protein kinase C (PKC).5 The use of a classical Y-27632 mw PKC activator, phorbol 12-myristate 13-acetate (PMA), in Huh7 cells could mimic the effect of 5HT in serum-free culture conditions, indicating that class 2 receptors are potentially involved in 5HT-mediated cell survival (Supporting Fig. 2B). After administration of specific agonists for the receptors 1/7 (5-CT), 2A (DOI), and 2B (α-Me-HTP), we concluded that the 2B receptor is responsible receptor for 5HT-mediated cytoprotection. This finding was further supported with ritanserin, a general 5HT2-receptor antagonist.

Ritanserin was able to reverse the cytoprotection conferred by 5HT, whereas antagonists targeted for the 5HT-1A, -2A, and -7 receptor 上海皓元医药股份有限公司 failed to provide an effect on cell viability (Supporting Fig. 2C). The presence of HTR2B was demonstrated by western blotting (Supporting Fig. 2D). In time-dependent experiments 5HT caused significantly higher values of MTT activity in Huh7 and HepG2 cells after 120 hours of serum deprivation compared to untreated cells (Fig. 2A,C). The same effect was also found with α-Me-HTP, a selective 5HT2B agonist17, 18 (Fig. 3B,D). When SB204741 (SB204), a selective 5HT2B antagonist,17, 18 was added during incubation with 5HT or α-Me-HTP the effect was abolished in a dose-dependent manner (Fig. 2A-D). Taken together, these experiments identified (1) the 5HT2B receptor as a mediator of survival, and (2) demonstrated that 5HT mediates proliferation of HCC cells as a result of cytoprotection. The combined Hoechst/TUNEL and calcein/ethidium stainings (Fig. 1A) suggested either necrotic or apoptotic cell death after serum deprivation. Therefore, we measured caspase-3 activity in Huh7 under different conditions to elucidate the cytoprotective mechanism of 5HT. Interestingly, serum deprivation was not associated with any detectable caspase-3 activity (Fig. 3A).