Tissue samples, Ethical Statement and Immunohistochemistry (IHC)
An OC tissue chip, containing 70 OC tissues and 10 normal tissues (#OVC1504) was purchased from the shanghai Superbiotek Pharmaceutical Technology Inc. (Shanghai, China). The study was approved by the Academic Ethics Committee of the Second Xiangya Hospital of Central South University and performed under the instructions of Declaration of Helsinki. IHC and scoring were applied according to our previous description  with primary antibody UBE2T (dilution: 1:50, BBI, Shanghai, China).
Normal ovarian epithelial cell line，IOSE80, and OC cell lines, SKOV3 and OVCAR3, were purchased from the American Type Culture Collection (ATCC, VA, USA). SKOV3 was maintained in RPMI-1640 medium plus 10% fetal bovine serum (FBS, Thermo, MA, USA). OVCAR3 and IOSE80 were cultured in DMEM medium plus 10% FBS (Thermo, MA, USA). The cells were culture in a humidified incubator at 37℃ and 5% CO2. The chloroquine (CQ, Sigma-Aldrich, MO, USA) and MK2206 (Selleck, TX, USA) were added into the culture medium for functional experiments as indicated in figure legends.
Small interfering RNAs (siRNAs) and plasmids transfection
The UBE2T siRNA (si-UBE2T) and scrambled siRNA (si-NC) were purchased from RiboBio Inc. (Guangzhou, China). The UBE2T expression plasmid, pENTER-UBE2T, and the control plasmid, pENTER-vector, were obtained from Vigene Inc. (Jinan, China). The si-UBE2T sequences are 5’-GTCCTGGTTCATCTTAGTTAA-3’, which targets the 3’ untranslated region of UBE2T mRNA. The siNC sequences were not offered by the manufacturer. SKOV3 and OVCAR3 cells were transfected with si-UBE2T or co-transfected with siUBE2T and pENTER-UBE2T using LipofectamineTM 2000 (Thermo, MA, USA) according to our previous description .
RNA isolation and quantitative real-time PCR(qPCR)
The total RNA isolation and qPCR was carried out as previous description . The expressions of UBE2T was determined by qPCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. The primer sequences of UBE2T and GAPDH are as follows. UBE2T, Forward 5’-ATCCCTCAACATCGCAACTGT-3’, Reverse 5’-CAGCCTCTGGTAGATTATCAAGC-3’; GAPDH, Forward 5’-ATGGAGAAGGCTGGGGCTC-3’, Reverse 5'-AAGTTGTCATGGATGACCTTG-3’.
Cell growth assay
The cell growth assay was performed as previously described . The effects of UBE2T rescue and MK2206 addition on cell growth were reflected by inhibitory growth rate with si-NC group as control. The experiments were independently performed in triplicate.
Plate clone formation assay
Plate clone formation assay was carried out as previously described . The experiments were independently performed for three times.
Transwell invasion assay
Transwell invasion assay was performed as previous description . The experiments were independently performed for three times.
The western blot was performed as previous description . Primary antibodies, including UBE2T (BBI, Shanghai, China), p53(Santa Cruz, TX, USA), BECN1(Abclonal, Wuhan, China), p62(Abclonal, Wuhan, China), LC3A/B (Abclonal, Wuhan, China), p-AKT(S473) (CST, MA, USA), AKT(CST, MA, USA), mTOR (CST, MA, USA), p-mTOR(S2448) (CST, MA, USA), β-actin (Proteintech, Wuhan, China), were used to detect their levels under specific treatments.
Statistical analyses and statistical charts were analyzed and produced using SPSS20.0 software and GraphPad Prism version 8. For comparisons between two groups, a Student t-test or chi-square test was carried out. Survival curves were obtained via Kaplan-Meier method, and the statistical analysis was evaluated by Log-rank test. The univariate and multivariate Cox regression was performed to analyze the relationship of among UBE2T expression, clinicopathological parameters, and survival in OC patients. For all analyses, P (two side) <0.05 was considered statistically significant.