Abstract: CHEN Xu*, GUO Xin-yun, XU Song, ZHANG Meng-li, JIN Hui, XING Mei-chun, HUANG Dan, REN Fa-liang, DU Kai-he, JU Mei, LI Xin-yu, CHEN Kun, ZHOU Zhi-hai, GU Heng. *Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: JU Mei, Email: jumeiweng@163.com; GU Heng, Email: guheng@aliyun.com 【Abstract】 Objective To evaluate the regulatory effect of serum starvation on autophagy in human HaCaT keratinocytes, and to investigate its molecular mechanism. Methods HaCaT cells were cultured either in Dulbecco′s modified Eagle′s medium （DMEM） + 10% fetal calf serum （control group） or in DMEM without fetal calf serum （starvation group）, for four hours. Then, cell appearance was observed by light microscopy, methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate cell viability, transmission electron microscopy to observe ultrastructural changes, monodansylcadaverine （MDC） staining to identify the formation of autophagosomes, and Western blot to measure the expression of microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ, LC3-Ⅰand autophagy related protein 7 （ATG7）. Additionally, the phosphorylation level of mammalian target of rapamycin （mTOR） at Ser2448 and Ser2481 was semiquantitatively analyzed by Western blot. Results Serum starvation increased the viability of HaCaT cells. Electron microscopy and MDC staining confirmed the formation of autophagosomes in serum-starved HaCaT cells. Compared with the control cells, the serum-starved HaCaT cells showed a significant increase in the transformation of LC3-Ⅰto LC3-Ⅱ （LC3-Ⅱ/LC3-Ⅰratio: 3.508 ± 0.415 vs. 0.538 ± 0.038, t = 13.32, P < 0.01）, and expression of ATG7 （ATG7/GAPDH ratio: 0.048 ± 0.011 vs. 0.021 ± 0.006, t = 7.27, P < 0.05）. The phosphorylation level of mTOR at ser2448 and ser2481 was significantly lower in the serum-starved HaCaT cells than in the control cells （phosphorylated mTOR（ser2448）/mTOR ratio: 0.394 ± 0.048 vs. 0.762 ± 0.108, t = 7.58, P < 0.05; phosphorylated mTOR （ser2481）/mTOR ratio: 0.111 ± 0.020 vs. 0.263 ± 0.039, t = 13.77, P < 0.05）. Conclusion Serum starvation can induce autophagy in and increase the viability of HaCaT cells, likely by inhibiting the activation of mTOR. 【Key words】 Keratinocytes; Autophagy; Cells, cultured