Fig. 3

STAT3 overexpression is protective against isoflurane-induced cytotoxicity in U251 cells. Western blot (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) were set as controls. Empty, empty vector-transfected cells. (C) Western blot analysis were performed after isoflurane exposure in U251 cells with or without STAT3 overexpression (n = 4). STAT3 overexpression was able to restore the decline 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) induced by isoflurane. The mitochondria-located STAT3 (mSTAT3) was also decreased after isoflurane exposure (n = 4, P = 0.003). STAT3 overexpression prevented this reduction (P < 0.001) and the release of cytochrome C (cytoC) from mitochondria into the cytoplasm after isoflurane exposure (P < 0.001). GAPDH was used as loading controls for whole cell as well as cytosolic fraction. Cytochrome C oxidase IV was used as loading controls for mitochondrial fraction. The blots were cropped according to the ladders and then incubated with different antibodies. The contrast and brightness of the blot images were adjusted uniformly. # P < 0.05, ## P < 0.01 versus control. * P < 0.05, ** P < 0.01 versus isoflurane only