STAT3 Involved in Cellular Vulnerability to Isourane

STAT3 signaling is crucial during neural spontaneous death period, a restricted developmental time window in which the neonatal brain is vulnerable to isourane. Here, we designed experiments to assess whether isourane target STAT3 to deliver its cytotoxicity. Mice at postnatal day 7 or 21, primary cortical neurons cultured for 5 or 14 days and human neuroglioma U251 cells were treated with isourane. A plasmid containing human wild-type STAT3, STAT3 anti-sense oligonucleotide, STAT3 specic inhibitor STA21, proteasome inhibitor MG-132 and calcineurin inhibitor FK506 were utilized to evaluate the inuence of STAT3 levels on isourane-induced cytotoxicity. In the present study, an upregulation of STAT3 parallel with a decline in calcineurin activity as well as a decrease in the ability of isourane to trigger calcineurin activity and neuroapoptosis were observed in more mature neuron or brain. STAT3 survival pathway was impaired after isourane exposure in U251 cells and exerted a prominent effect. STAT3 disruption exaggerated isourane-induced oxidative injury and apoptosis, whereas, STAT3 overexpression exhibited notable cellular protection. The blockage of calcineurin activity ameliorated neural apoptosis, dendritic spine impairment and cognitive dysfunction induced by isourane. Overall, these results indicated that specic regulation of STAT3 was closely related with the cellular vulnerability to isourane.


Introduction
Accumulating evidence suggests that most commonly used clinical anesthetics causes widespread neuronal apoptosis in newborn animals [1,2], whereas the mature brain appears resistance [3,4]. Animal research further showed that anesthetics may trigger cell death in immature neurons of a speci cally vulnerable age, when they require synaptic connections and neurotrophin such as NGF and BDNF for survival [5]. However, the underlying mechanism of this inherent vulnerability is largely unknown.
As a transcriptional factor and an intracellular signal transducer, signal transducer and activator of transcription-3 (STAT3) is increasingly recognized for its neuroprotective effect in various brain injuries [6]. During postnatal brain development, STAT3 is identi ed as a key mediator of the neurotrophininduced survival pathway, and its protein level gradually increases as neurons pass through the developmental death period [7]. STAT3 knockdown in mature neurons induces neurotrophin dependency, whereas overexpression of STAT3 enables immature neurons to achieve resistance against neurotrophin deprivation.
Our previous study found that iso urane impaired the STAT3-mediated survival pathway in the brain of neonatal mice. In the current study, we hypothesized that the impaired STAT3 pathway further sensitize cells to the toxicity of iso urane. We designed gain and loss experiments to determine whether STAT3 signaling was involved in the cellular vulnerability to iso urane.

Mice anesthesia and treatment
Animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and were approved by the Ethics Review Board for Animal Studies of Nanjing Drum Tower Hospital. The animal treatments were similar to that in our previous study [8]. C57/BL6 mice on postnatal day 7 or 21 (from Model Animal Research Center of Nanjing University, China) were anesthetized with 1.5% iso urane for 6 hr in 100% oxygen at 37°C. Mice in the interaction group were injected intraperitoneally with a calcineurin inhibitor FK506 (5 mg/kg, InvivoGen, USA) 30 min before iso urane exposure.
Primary neuron culture and treatment The primary cortical neurons were cultured in vitro for 5 days (C5) or 14 days (C14) and treated with 1.5% iso urane in a gas mixture of 21% O 2 , 5% CO 2 , and balanced nitrogen for 6 hr as we described previously [9]. In the interaction experiments, neurons were pretreated with FK506 (1mM) 30 min before iso urane exposure.

U251 cell culture and treatment
We used U251 human neuroglioma cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) in the succeeding experiments. Cells incubated in serum-free media were exposed to 2% iso urane for 6 hr, performed similarly as our previous study [8]. The wild-type pcDNA3-6×Myc-mSTAT3 vector [10] or an antisense oligonucleotide (ASO) of STAT3 (HSS186131, Invitrogen, USA) were transfected into cells using lipofectamine TM 2000 (Invitrogen, USA). An AxyPrep Endofree Plasmid Miniprep Kit (Axygen Biosciences, USA) was used to extract the plasmids from bacterial culture. After the transfection, cells were incubated at 37°C in a CO 2 incubator for 48 hr prior to iso urane exposure. The untransfected cells without iso urane exposure and the empty vector-transfected cells were set as the controls. In the interaction studies, cells were pretreated with a proteasome inhibitor MG-132 (30mM, Calbiochem, Germany) [11], FK506 (1mM) or a STAT3 inhibitor STA21 (30mM, Enzo Life Sciences, Switzerland) [12] 30 min before iso urane exposure.

Mitochondria isolation
A mitochondria isolation kit for cultured cells (Thermo Scienti c, USA) was used to evaluate the levels of mitochondria-located STAT3 and cytochrome C oxidase released from mitochondria into cytosol in U251 cells. After exposed to iso urane, cells grown on 10-cm culture dishes (appropriately 1 ´ 10 7 cells incubated in 9 ml of culture media per dish) were collected, centrifuged at 850×g for 2 min, and supernatant was then discarded. Subsequently, 800 μL of mitochondria isolation reagent A (with 100:1 protease inhibitors), provided in the kit, was added to each sample. The resulting mixtures were incubated on ice for exactly 2 min. Option A, which used the reagent-based method, was utilized in accordance with the manufacturer's protocol.
Real-time PCR (RT-PCR) U251 cells (n = 4 wells) grown in six-well plates were treated with lysis buffer provided by an Rneasy Mini Kit (QIAGEN, Hilden, Germany) immediately after iso urane treatment. The frontal cortex was harvested, frozen in liquid nitrogen, and stored at −80°C before use. The tissues (n = 4) were ground in liquid nitrogen using a small mortar and then treated with lysis buffer. Retrotranscription and PCR analysis were carried out as we described previously [9]. STAT3, cyclin D1, Mcl-1, survivin, and Bcl-xl genes (3 repetitions per sample) were ampli ed using speci c oligonucleotide primers (GenScript, USA; see table, Supplemental Digital Content , which is a table listing sequences of all primers used in this study). Housekeeping gene, b-actin, was used as internal standard. The no template sample was used as negative control.

Western blot analysis
The brain tissues were immediately removed and stored in -80℃ after anaesthesia . The frontal cortex were homogenized with lysis buffer containing 1% protease and phosphatase inhibitor cocktail (Thermo scienti c, USA) on ice for 30 min. For the in vitro experiments, lysis buffer was added to six-well plates seeded with U251 cells or primary neurons immediately after 6-h iso urane exposure. Protein concentration was measured using a BCA protein assay kit (Thermo Scienti c, USA) and then each

Detection of intracellular ROS production
A uorescent probe carboxy-H2DCFDA (Invitrogen, Eugene, USA) was used to assess the levels of intracellular reactive oxygen species (ROS). U251 cells grown in six-well plates were incubated with ROS detection solution for 30 min at 37 °C in the dark (n = 6 wells). Each sample was then washed thrice with Hank's buffer and immediately observed under a Fluoview Fv10i confocal microscope (Olympus, Japan). A uorescence spectrophotometer (F-7000, Hitachi, Japan) was also used to measure the cellular uorescence of cells grown on 96-well plates (n = 6 wells).

Flow cytometric detection of apoptosis
A FITC Annexin V apoptosis detection kit (BD Biosciences, USA) was used to quantify the apoptotic rate in U251 cells (n = 6 wells). Cells grown on six-well plates were collected, washed twice with cold PBS, resuspended in 1 ´ binding buffer at a concentration of 1 ´ 10 6 cells/ml, and then incubated with 5 μL FITC Annexin V as well as 5 μL PI for 15 min in the dark. The apoptotic rate was analyzed by a ow cytometer (BD Biosciences, USA) and expressed as the percentage of FITC Annexin V positive cells out of the total number of cells counted.
Immuno uorescence analysis U251 cells or primary neurons grown on six-well plates were xed in 4% PFA for 30 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% BSA for 1 hr (n = 3). Rabbit primary antibodies against STAT3 (1:100), Drebrin (1:100; Abcam) and Alexa Fluor 488 donkey anti-rabbit secondary antibodies (1:200; Molecular Probes, USA) were used for immuno uorescence analysis. Images (n = 3) were captured using a Fluoview Fv10i confocal microscope (Olympus, Japan) and analyzed using Image-Pro Plus analysis software (Media Cybernetics, Inc., Rockville, MD). TUNEL staining U251 cells or primary neurons grown on six-well plates were assayed through TUNEL staining by using the in situ cell death detection kit (Roche Applied Science, Germany) per the manufacturer's protocol. After xed in 4% PFA for 1 hr, cells were permeabilized with 0.1% Triton X-100 for 2 min on ice and stained with TUNEL for 1 hr at 37 °C in the dark. Representative images were obtained at 200´ magni cation by using a TCS SP2 multiphoton confocal microscope (Leica, Germany).

Calcineurin activity assays
The activity of calcineurin was determined in the frontal cortex obtained from mice that were killed immediately after anesthesia or age-matched controls (n = 6). Fresh tissues were homogenized with lysis buffer containing 1% protease inhibitor (Thermo scienti c, USA). A colorimetric calcineurin cellular activity assay kit (Calbiochem, Germany) was used to perform the test as we previously described [9].

Intellicage test
Mice assigned for behavior assessment were allowed to recover in 100% oxygen for 20 min after iso urane exposure and reunited with their mothers. Mice were housed in a temperature-controlled (21± 1°C ) room with a 12-h light/dark cycle and free access to food and water. Four weeks after iso urane exposure, place learning was performed in the IntelliCage system (NewBehavior Inc., Zurich, Switzerland) as we described before [8]. Brie y, the test started from an adaptation period of 6 days with ad libitum access to obtain water in all the corners. The least preferred corner for each mouse was chosen, and for the following 4 days, mice could obtain access to water only in their assigned correct corner. A brief air puff (1 bar/2 s) was triggered as a punishment if the mice visit an incorrect corner. When the test turned to the reversal learning phase, the opposite corner to the initial correct corner was reassigned as correct corner to each mouse for the next 4 days. Place learning was assessed as a ratio of number of visits in correct corner to all corners.

Statistics
Data are expressed as mean ± SD. The Western blot results, levels of mRNA and intracellular ROS, apoptotic rate, and activity of calcineurin were analyzed using one-way ANOVA for overall differences among groups followed by Bonferroni post hoc test for multiple comparisons. Unpaired Student's t-test was used for comparisons between 2 groups. Repeated measures ANOVA followed by Bonferroni post hoc test was used to examine statistical comparisons among the four groups on correct visit ratios at different time point in Intellicage test. The sample sizes used were based on previous experience. All analyses were performed using SPSS 13.0 software (IBM Corporation, Armonk, USA). P < 0.05 (2-tailed) was considered as signi cant difference.
Iso urane exposure impaired STAT3 survival pathway in U251 cells To determine whether STAT3 is crucial in cellular vulnerability to iso urane, we tested the role of ectopic STAT3 in iso urane-induced apoptosis. We employed U251 human neuroglioma cells, which showed caspase-3 activation and ROS accumulation after a 6-h iso urane exposure in our previous study. The present study showed that decreased protein levels of STAT3, pY705-STAT3 and its downstream survival targets were observed in U251 cells after iso urane exposure (Figure 2A; n = 4, P < 0.001 for STAT3 and pY705-STAT3, P = 0.001 for Bcl-xl and survivin). Immuno uorescence showed that iso urane exposure induced a 30% reduction of STAT3 staining ( Figure 2B; n = 6, P < 0.001) in U251 cells, indicating that this cell model may be used for the study of STAT3 function in iso urane-induced cytotoxicity in vitro.
In contrast with the reduction of STAT3 protein, the mRNA level of STAT3 in U251 cells was increased remarkably after 6-h iso urane exposure ( Figure 2C; n = 4, 2.34 ± 0.14 vs 1.04 ± 0.08, P < 0.001), indicating that iso urane-induced STAT3 reduction may be caused by a post-transcriptional mechanism. Protein degradation is known to be achieved mostly through the ubiquitin (Ub)-proteasome pathway (UPP) [14]. To determine the role of STAT3 degradation in iso urane, a proteasome inhibitor MG132 was introduced ( Figure 2D). Interestingly, our data showed that iso urane signi cantly decreased the levels of total ubiquitinated proteins in U251 cells (n = 4, P < 0.001). MG132 prevented this downregulation of ubiquitinated proteins (P < 0.001), so as STAT3 (P = 0.012). However, MG-132 coincubation did not lead to any further enhancement of protein ubiquitination (P = 0.14), indicating that iso urane accelerated the degradation of ubiquitinated proteins, but not protein ubiquitination. Moreover, this MG132 effect enhanced the levels of Bim (P < 0.001), which is an essential initiator of apoptosis, with an increased caspase-3 activation (P < 0.001) after iso urane treatment. In contrast, pretreatment with FK506, a calcineurin-speci c inhibitor, prevented the degradation of STAT3 ( Figure 2E, n = 4; P < 0.001) and cleavage in caspase-3 (P < 0.001), without signi cant in uence to the decreased levels of total ubiquitinated proteins (P = 1.0) after iso urane exposure. These ndings highlighted the prominent importance of STAT3 in iso urane-induced cytotoxicity.
Next, we found that STAT3 overexpression was able to restore iso urane-induced decline of STAT3 and its downstream anti-apoptotic proteins ( Figure 3C; n = 4, P = 0.011 for Mcl-1, P = 0.006 for survivin) as well as mitochondria-located STAT3 ( Figure 3D; n = 4, P < 0.001), which has been previously con rmed to play a major role in modulating mitochondrial respiration and antioxidative stress [15]. Simultaneously, cytochrome c that released from mitochondria into cytoplasm after iso urane exposure was also prevented ( Figure 3D; n = 4, P < 0.001).
The protective effects of STAT3 involved an antioxidative stress mechanism ROS accumulation was reported to be a critical event in triggering the cytotoxicity of iso urane [16]. To determine whether the cellular protective function of STAT3 involved an antioxidative stress mechanism, we used an antisense oligonucleotide (ASO) to knockdown STAT3 and a speci c inhibitor STA21 to hinder STAT3 dimerization, together with the STAT3 overexpression assay. Western blot and immuno uorescence analyses showed that STAT3-ASO e ciently knocked down STAT3 expression in U251 cells ( Figure 3A; n = 3, P < 0.001), meanwhile 6-h STA-21 treatment markedly inhibited the nuclear translocation of STAT3, without signi cant in uence on its total protein level. STAT3 knockdown or its nuclear-translocation disruption resulted in a more than 10-fold increase of ROS in U251 cells after iso urane exposure, when compared with that of controls ( Figure 3B; n = 6, P < 0.001). The apoptotic rates after iso urane exposure ( Figure 3C & D; n = 6, 12.42 ± 2.23%) were obviously augmented in cells with STAT3 knockdown (24.78 ± 4.65%, P < 0.001) or STA21 treatment (19.10 ± 3.31%, P < 0.001). By contrast, STAT3 overexpression mitigated iso urane-induced ROS accumulation ( Figure 3B; n = 6, P < 0.001) and apoptosis ( Figure 3C & D; n = 6, 7.51 ± 1.33%, P = 0.002). These protective effects of STAT3 were also con rmed by TUNEL staining (Figure 3E; 3 wells per group, 6 images per well) and Western blot analysis of cleaved caspase-3 ( Figure 3F & G; n = 4). Notably, STAT3 disruption or overexpression did not affect the levels of ROS and apoptosis in iso urane-untreated cells.
Mechanistically, the antioxidative effect of STAT3 was linked to its canonical activity as a nuclear transcription factor. We found the protein level of manganese-containing superoxide dismutase (MnSOD or SOD2), a critical cellular antioxidant enzyme and a direct target of STAT3 [17,18], was decreased ( Figure 3F & G; n = 4, P < 0.001) after iso urane exposure. The decline was further aggravated in cells with STAT3 disruption, but was restored by STAT3 overexpression (P < 0.001), suggesting that iso uraneinduced ROS accumulation is at least partially ascribed to impaired ROS scavenging.

Calcineurin inhibition alleviated the neurotoxicity of iso urane
Since STAT3 levels were closely related with cellular vulnerability to iso urane, we next examined whether calcineurin-speci c inhibitor FK506 have a long-term protective effect against the neurotoxicity of iso urane. STAT3 degradation ( Figure 4A; n = 4, P < 0.001) and apoptosis ( Figure 4B; 4 wells per group, 6 images per well) induced by iso urane were prevented by FK506 pretreatment in C5 neurons. After exposed to iso urane, C5 neurons were maintained in culture for 5 days and then dendritic spines were stained using a neuronal F-actin marker, drebrin. Some recent studies on neurological disorders accompanied by cognitive de cits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction [19]. In the present study, we observed that neurons in the control group exhibited extensive and overlapping neurites. A signi cant reduction in the number of dendritic spines was detected in neurons exposed to iso urane. Pretreatment with FK506 attenuated the iso urane-induced loss of dendritic spines ( Figure 4C; 4 wells per group, 6 images per well).
The post-transcriptional mechanism was also involved in iso urane-induced STAT3 downregulation in the brain of P7 mice. The protein levels of STAT3 were not signi cantly decreased until iso urane exposure for 4 hr ( Figure 4D; n = 4, P < 0.001). In contrast, the mRNA level of STAT3 was increased remarkably after 1-h iso urane exposure ( Figure 4E; n = 4, P < 0.001). Intriguingly, the protein level of STAT3 was elevated initially, and the underlying mechanism remained to be determined.
Finally, we used the Intellicage system to assess the long-term effect of FK506 on iso urane-induced cognitive dysfunction. As we described before, the cognitive impaired induced by iso urane exposure was displayed in a more demanding task. During the rst three days of learning phase, the iso urane-treated mice did not show a signi cant disability in recognizing the correct corner. When test turned to the reversal learning phase, as compared to mice in the control group, the ratio for making a correct visit was approximately 20% lower in the iso urane-treated mice ( Figure 4F; n = 8, P < 0.01 at day 4-8), re ecting that they had not learned to drink successfully in their new assigned corner. Mice pretreated with FK506 showed signi cant improvement in spatial memory as compared to that in the iso urane group ( Figure  4F; n = 8, P < 0.01 at day 4-7, P < 0.05 at day 8).

Discussion
In the present study, we propose the hypothesis that STAT3 signaling is involved in the cellular vulnerability to iso urane. Our results showed that calcineurin-mediated degradation of STAT3 is closely related with the cytotoxicity of iso urane in the brain of mice and cultured primary cortical neurons. Subsequently, we used the U251 cell line to determine the underlying mechanism and found that STAT3 is of particular importance in iso urane-induced neurotoxicity. STAT3 disruption exaggerated, while STAT3 overexpression mitigated iso urane-induced oxidative injury and apoptosis.
Calcineurin is a calcium and calmodulin dependent serine/threonine protein phosphatase that is highly expressed in brain [20]. Recently, it has also been shown that calcineurin promotes the degradation of STAT3 and controls the critical developmental death of neurons in newborn brain [21]. The present study found that the iso urane exposure resulted in a 2-fold increase of calcineurin activity in the frontal cortex of P7 mice, but not in P21 mice. Moreover, the calcineurin activity of P21 mice was signi cantly lower than that of P7 mice, accompanied by a marked elevation in STAT3 protein levels in the frontal cortex. A similar developmental regulation of STAT3 was exhibited in primary cortical neurons cultured in vitro. Compared with C14 neurons, an upregulation of STAT3 parallel with a decline in calcineurin activity as well as with a decrease in the ability of iso urane to trigger calcineurin activity were observed in more mature C14 neurons. This nding revealed that calcineurin-mediated STAT3 degradation may be closely related with developmental stage-related vulnerability of the brain to iso urane stress. Given previous research showing that iso urane induce neurodegeneration by activating inositol 1,4,5-trisphosphate (IP3) receptors [22,23], it is possible that the upstream trigger of calcineurin activity could be the excessive release of calcium from the endoplasmic reticulum.
The ubiquitin-proteasome pathway is the major intracellular non-lysozymal mechanism for degrading proteins and is involved in the regulation of a number of key signaling pathways. Proteins destined for degradation are conjugated by an ubiquitin chain and the ubiquitinated proteins are subsequently targeted for degradation by proteasome [24]. Previous studies have shown that ubiquitin metabolism affects growth inhibition of iso urane in yeast [25,26]. A recent research revealed that iso urane preconditioning decreased ubiquitin-conjugated protein aggregation and prevented free ubiquitin depletion in the CA1 region of hippocampal after global cerebral ischemia in mice [27]. It is noteworthy that we found iso urane exposure triggered a 40% decrease in total ubiquitin-conjugated proteins levels in U251 cells, while when co-incubation with a proteasome inhibitor MG132, the ubiquitin-conjugated proteins levels were not increased, indicating that iso urane accelerated the degradation of ubiquitinconjugated proteins, but not promote ubiquitin conjugate to protein. In addition, our study found that MG132 result in aggravated apoptosis after iso urane exposure, hence it is essential to alternative techniques to speci cally regulate ubiquitination and proteasomal degradation of the key protein.
As a transcription factor, STAT3 is suggested to play an instructive role in brain development. STAT3mediated cytokine signaling regulates gliogenesis as well as neurogenesis during brain development [28]. Neurons in newborn brain are under selection through a process of developmental death, depend on neurotrophin for survival. Overexpression of STAT3 completely attenuated neurotrophin dependency in this state, suggesting STAT3 is a key mediator of survival pathway that neurons acquire in this vulnerable period [29]. Moreover, it has been reported that calcineurin mediate STAT3 degradation via directing STAT3 to the E3 ubiquitin ligase complex [21]. To identify whether STAT3 is a key mediator of the cellular vulnerability to iso urane, we performed gain-and loss-of-function studies in U251 cells. We con rmed that the levels of STAT3, p-STAT3 and its downstream anti-apoptotic proteins were signi cantly reduced after iso urane exposure in U251 cells. In comparison with global blockade the degradation of ubiquitinated proteins by MG132 aggravated apoptosis after iso urane exposure, speci cally blocking the degradation of STAT3 by calcineurin-inhibitor FK506 prevented caspase-3 activation after iso urane exposure, revealing the particular importance of STAT3 in iso urane-induced cytotoxicity.
This notion was further con rmed by the result that STAT3 overexpression mitigated iso urane-induced ROS accumulation, caspase-3 activation and apoptosis in U251 cells, whereas cells with STAT3 knockdown exhibited more sensitive to the toxicity of iso urane, mimicking the cellular phenomenon of immature neurons with relatively lower levels of STAT3. Moreover, STAT3 knockdown or overexpression did not show signi cant in uence on the basal levels of ROS and apoptotic rate in iso urane-untreated cells, suggesting that STAT3 was functioning in a stress-related protective mechanism.
Oxidative injury was regarded as a key event in iso urane-induced cell apoptosis [30,31]. Previous studies have reported both enhanced ROS production [32] and impaired ROS scavenging were involved in the ROS accumulation after iso urane exposure [33]. It has been demonstrated that iso urane impaired SOD but not catalase activity, while it remains unclear whether the impaired ROS scavenging capability is an initial event or a result of excess ROS accumulation. ROS is generated when electrons released from the mitochondrial electron transport chain (ETC) incompletely reduce O2, mainly form complexes I and III. STAT3 has recently been identi ed to colocalize with complex I and shown essential for optimal function of the electron transfer chain [15,34,35]. Mitochondrial STAT3 appeared to exert protection against certain diseases [18,36,37] by inhibiting oxidative stress or mitochondrial permeability transition pore (mPTP) opening [38,39]. Notably, the present study showed that mitochondria-located STAT3 was also decreased after iso urane exposure and was able to be restored by STAT3 overexpression. Meanwhile, we found that inhibiting the nuclear translocation of STAT3 by STA21 greatly aggravated iso urane-induced ROS accumulation and apoptosis as well as STAT3 knockdown. Thus, STAT3 overexpression may contribute to cytoprotection not only through its transcriptional activity such as upregulating anti-oxidative and antiapoptotic proteins, but also through its mitochondrial function that reducing ROS generation.
Taken together, the present study demonstrated that STAT3-mediated survival pathway as well as its antioxidative effect were impaired after iso urane exposure and contributed to the cellular vulnerability to iso urane. The connections between calcineurin-mediated STAT3 degradation and neuronal vulnerability to iso urane reported here point out novel therapeutic target for the prevention and treatment of iso urane-induced neurotoxicity, not only to the immature brain, but also the aged brain, in which a marked decrease of STAT3 was also reported [40]. In addition to STAT3, a marked decrease in the amount of ubiquitin-conjugated proteins protein was found after iso urane exposure. Continued analysis of additional proteins relevant to iso urane-response degradation is imperative in future studies.

Con ict of Interest
The authors have no relevant nancial or non-nancial interests to disclose.

Availability of data and material
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Code availability
The software application and custom code are available from the corresponding author upon reasonable request.

Ethics approval
Animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Review Board for Animal Studies of Nanjing Drum Tower Hospital.

Consent to participate
Not applicable.

Consent to publication
Not applicable.

Figure 2
Iso urane exposure impaired STAT3 survival pathway and ubiquitinated protein degradation in U251 cells. (A) The protein levels of STAT3, pY705-STAT3 and its downstream Bcl-xl and survivin were signi cantly declined (P < 0.001 for STAT3 and pY705-STAT3, P = 0.001 for Bcl-xl and survivin, n = 4) in U251 cells after a 2% iso urane exposure for 6hs. The levels of protein were expressed as optical density normalized to housekeeping gene GAPDH. (B) Immuno uorescence analysis detected a notable reduction Overexpression of STAT3 is protective against iso urane-induced cytotoxicity in U251 cells. Western (A) and RT-PCR (B) analysis showed that the transcription and translation of STAT3 as well as its target genes (n = 4) were remarkably upregulated after the transfection of STAT3-pcDNA3 into U251 cells (STAT3). The untransfected cells (Ctrl), and the empty vector-transfected cells (Empty) were set as controls. Representative immunoblotting images (C) and band density analyses (D) revealed that overexpression of STAT3 (n = 4) prevented the iso urane-induced decrease of STAT3 (P < 0.001), pY705-STAT3 (P < 0.001) and its downstream anti-apoptotic proteins (P = 0.011 for Mcl-1, P = 0.006 for survivin). The mitochondria-located STAT3 was decreased after iso urane exposure (n = 4, P = 0.003).
STAT3 overexpression restored this reduction (P < 0.001) and protected cytochrome c from releasing to cytoplasm (P < 0.001) that induced by iso urane. GAPDH was used as loading controls for whole cell as well as cytosolic fraction, and cytochrome c oxidase IV was used as loading controls for mitochondrial fraction. # P < 0.05, ## P < 0.01 versus control. * P < 0.05, ** P < 0.01 versus iso urane. mSTAT3 = mitochondria-located STAT3, cytoC = cytochrome c. Figure 5