carried out the experiments and performed the statistical analysis. Funding The authors declare that there are no sources of funding to be acknowledged.. I, II, and IV around the electron transport chain were significantly decreased under isoflurane treatments. Importantly, ovarian cancer cells become hypersensitive to glycolysis inhibitors with isoflurane pretreatments. The present study demonstrates that isoflurane treatments drive a metabolic switch of ovarian cancer cells and contributes to the discovery and development of clinical therapeutic brokers against ovarian cancer. or control antisense or pre-using Lipofectamine RNAi MAX reagent (Thermo Z-Ile-Leu-aldehyde Fisher Scientific, Waltham, MA) according to the manufacturers instructions. Seventy-two hours after transfection, cells were collected for the following experiments. RNA isolation and quantitative RT-PCR Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturers protocol. RNA (1 g) in each treatment was reverse transcribed into cDNA using iScript Reverse Transcription Supermix (BioCRad). For detection of miRNAs, the miScript SYBR Green PCR Kit (Qiagen, Shanghai, China) was used for the measurement of expressions. U6 small nuclear RNA was used as an internal control for for 15 min at 4C and the supernatants were collected. The lysates were separated by SDS/PAGE Z-Ile-Leu-aldehyde and then transferred on to the nitrocellulose membrane (BioCRad). Membranes were blocked with 5% nonfat milk in PBS made up of 0.1% Tween-20 (PBS-T) for 1 h at room LEPR temperature, the membranes were incubated with antibodies against GLUT1, PKM2, HK2, LDHA (Glycolysis Antibody Sampler Kit #8337, Cell Signaling, Danvers, MA, U.S.A.) and -actin (#4970, Cell Signaling, Danvers, MA, U.S.A.) at 1:1000 dilution at 4C overnight. Membranes were washed three times and incubated with secondary antibody (IRDye conjugated IgG, LI-COR) in PBS-T made up of 5% nonfat milk for 1 h. The signals were then detected with Odyssey Imaging System (LI-COR). Statistics analysis Statistical analyses were performed using GraphPad Prism 5.0. The statistical significance was decided with a two-tailed Students test for unpaired data. in SKOV3 and TOV21G cells under isoflurane treatments since recent publications described that isoflurane could induce the expression in cardiomyocytes [9,15]. As we expected, is significantly up-regulated by isoflurane treatments (Physique 3A), suggesting that might involve in isoflurane-regulated glycolysis in ovarian cancer cells. We next measured the AKT pathway which has been reported to positively regulate glycolysis [16]. Results in Physique 3B illustrated that isoflurane could induce the phosphorylation of AKT. We transfected into ovarian cancer cells (Physique 3C) and found that overexpression of significantly up-regulated Akt phosphorylation (Physique 3D). The glycolysis enzymes HK2, PKM2, and LDHA were significantly up-regulated at protein levels and mRNA levels by overexpression (Physique 3D,E). Moreover, inhibition of suppressed both basal level and isoflurane induced glycolysis enzymes expressions (Physique 3F,G), indicating that isoflurane-promoted glycolytic rate was through up-regulation of expression and phosphorylation of AKT(A) SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 2 h, and the expressions of were assessed by qRT-PCR. U6 was used as an internal control. (B) SKOV3 and TOV21G cells were treated with isoflurane for 1 or 2 2 h, the phosphorylation of AKT was measured by Western blot. -actin was used as the loading control. (C) SKOV3 and TOV21G cells were transfected Z-Ile-Leu-aldehyde with control miRNAs or pre-for 72 h. The expression of was assessed by qRT-PCR. U6 was used as the internal control. (D) The proteins and (E) mRNAs of HK2, PKM2, and LDHA were measured in SKOV3 and TOV21G cells without or with overexpression. (F) SKOV3 and TOV21G cells without or with isoflurane treatment were transfected with control antisense or anti-for 72 h. The expression of was assessed by qRT-PCR. U6 was used as an internal control. (G) The mRNAs of GLUT1, HK2,.
carried out the experiments and performed the statistical analysis
