Fig. 1

STAT3 degradation mediated by calcineurin was closely related with the neural vulnerability to isoflurane. (A) The protein levels of STAT3 in the frontal cortex of mice on postnatal day 7 were assessed after isoflurane exposure for varying durations (n = 4). GAPDH was used as an internal control. ## P < 0.01 versus control. The polyvinylidene difluoride membranes with transferred proteins were cropped according to the protein ladders and then incubated with different antibodies. The contrast and brightness of the blot images were adjusted uniformly. (B) The mRNA levels of STAT3 in the frontal cortex of mice on postnatal day 7 were assessed after isoflurane exposure for varying durations by Real-Time PCR (n = 4). ## P < 0.01 versus control. (C) Isoflurane significantly increased the level of activated caspase-3 (n = 4, P < 0.001) in the frontal cortex of mice on postnatal day 7 (P7), but not in mice on postnatal day 21 (P21) (n = 4, P = 0.96). The protein levels of STAT3 were significantly higher in P21 mice compared to P7 mice (P < 0.001). The levels of caspase-3 activation were assessed by quantifying the ratio of cleaved and full-length caspase-3 first and then compared with that of control conditions. ## P < 0.01 versus P7 control mice. (D) The activity of calcineurin in the frontal cortex of P21 mice was significantly lower than that in P7 mice (n = 6, P < 0.001). Isoflurane increased calcineurin activity (n = 6, P < 0.001) in the frontal cortex of P7 mice, while the increase in P21 mice was minimal (n = 6, P = 0.21). The activity of calcineurin was expressed in nanomolar PO₄ per milligram of protein. ## P < 0.01 versus control in P7. (E) The primary cortical neurons were cultured in vitro for 5 days (C5) or 14 days (C14) prior to experiments. A similar stage-dependent proapoptotic effect of isoflurane accompanied by a developmental regulation of STAT3 was also exhibited in neurons (n = 4). ## P < 0.01 versus C5 control neurons. (F) The activity of calcineurin in C14 neurons was significantly lower than that in C5 neurons (n = 6, P < 0.001). No significant changes were observed in C14 neurons after isoflurane exposure (n = 6, P = 0.76). ## P < 0.01 versus C5 control neurons. Ctrl, control; Iso, isoflurane; T-Csp3, full-length caspase-3; Cl-Csp3, activated caspase-3