Fig 1: NVP-BEZ235 and rapamycin repressed the PI3K/AKT/mTOR pathway. SUNE1 cells were treated with 100 nM NVP-BEZ235, 100 nM rapamycin, or 100 nM NVP-BEZ235 and 100 nM rapamycin for 48 h. The control group was administered the same amount of vehicle. Total RNA from the four groups was isolated for reverse transcription-quantitative polymerase chain reaction. The mRNA levels of (A) PI3K, (B) AKT and (C) mTOR were normalized to the internal control GAPDH. (D) The protein levels of PI3K, AKT, p-AKT, mTOR and p-mTOR were detected by western blotting. (E-K) The protein density of PI3K, AKT, p-AKT, mTOR and p-mTOR was calculated relative to the internal control GAPDH, and the p-AKT/AKT and p-mTOR/mTOR ratios were calculated. **P<0.01 vs. control. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; p, phosphorylated; NVP, NVP-BEZ235.
Fig 2: Blots of (A) anti-apoptotic and (B) pro-apoptotic proteins at 48 h. 7,8-Dihydroxycoumarin concentrations: 25, 50 and 100 µmol/l; blank: control cells; glyceraldehyde 3-phosphate dehydrogenase (GAPDH): housekeeper gene; pAkt1, phospho-Akt1; pI?Ba, phospho-I?Ba.
Fig 3: How EEF1D regulates PI3K/OPTN/Akt signal pathway was explored by OPTN knockdown in SKOV3/DDP/KD cell line. Data are mean ± SD, the experiment was performed with sextuplicate in three independent sets, **P < 0.01 vs. scrambled shRNA or OPTN shRNA control, respectively; #P < 0.05 vs. scrambled shRNA in the same drug intervention
Fig 4: Reducing the expression of EEF1D gene regulated the contents and/or activities of proteins associated with PI3K/Akt signaling pathway, apoptosis and repair of DNA damage in xenografted tumor. A The related proteins in xenografted tumor tissues were detected with Western blotting, and ß-actin was used to show the similar amount of protein loaded in different lanes. B, D Relative intensities of protein bands in A were determined using Quantity-One software and normalized using ß-actin band intensity. C Akt phosphorylation levels were calculated according to the relative intensities of p-Akt/Akt bands, normalized using ß-actin. Data in B, C and D are presented as mean ± SD of six mice in each group. * P < 0.05, ** P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. DDP group
Fig 5: PI3K/Akt inhibitors confirmed that EEF1D could regulate PI3K/Akt signal pathway. A and B PI3K/Akt inhibitors would decrease viability of EEF1D knockout/knockdown cells less than control cells; A SKOV3/DDP cell lines, B SKOV3 cell lines. C and D PI3K/Akt inhibitors had the similar effects from knockout/knockdown cells; C SKOV3/DDP cell lines, D SKOV3 cell lines. Data are mean ± SD, the experiment was performed with sextuplicate in three independent sets, *P < 0.05, **P < 0.01 compared with DDP intervention alone in the same cell line; #P < 0.05, ##P < 0.01 compared with SKOV3 or SKOV3/DDP in the same drug intervention
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