Control (cells without propofol exposure). Activation of Nrf2 by propofol stimulation We then evaluated the effect of propofol stimulation on activation of Nrf2 in mRNA and protein levels. The results showed that 3-Methyladenine nmr exposing to propofol (20 μmol/L) for 48 h up-regulated the expression of Nrf2 at mRNAs levels (Figure 2A). Besides, exposing to propofol (20 μmol/L) for 48 h also up-regulated the protein expression of both HO-1 and Nrf2 (Figure 2B). Moreover, cells exposed

to propofol showed translocation of Nrf2 into the nucleus (Figure 2C). Figure 2 Activation of Nrf2 by propofol stimulation. (A) After stimulating by propofol, Nrf2 mRNA levels were quantified by real-time PCR analysis. Data were normalized by using GAPDH as an internal standard. * P < 0.05 vs. Control (cells without propofol exposure). These experiments were performed in triplicate. (B) After stimulating by propofol, HO-1 and Nrf2 protein level was analyzed by western blot. β-actin expression was monitored as the internal standard. (C) After stimulating by propofol, subcellular location of Nrf2 was detected by immunofluorescence assay. Propofol stimulation increased translocation of Nrf2 into the nucleus. Knock-down of Nrf2 by specific shRNAs In order to knock down Nrf2,

we constructed Talazoparib Nrf2-shRNA recombinant plasmids and transfected them into GBC-SD cells to knockdown the expression of Nrf2. qRT-PCR and western blot showed that Nrf2 expression was dramatically down-regulated at both the mRNA and protein levels Phosphoprotein phosphatase in GBC-SD cells compared with parental cells and Sh-NC (Figure 3A and Figure 3B). Among the four recombinant plasmids, ShRNA-1118 and ShRNA-2019 has the highest suppression efficiency, so both of them were used to process

the following experiments. Figure 3 Knock-down of Nrf2 by specific shRNAs. Forty-eight hours after transfection, cells were harvested. (A) Nrf2 mRNA levels were quantified by real-time PCR analysis. Data were normalized by using GAPDH as an internal standard. *P < 0.05 vs. Control (parental cells). (B) Nrf2 protein level was analyzed by western blot. β-actin expression was monitored as the internal standard. Loss of Nrf2 reverses the effects of propofol on cell proliferation, apoptosis, and invasion Finally, we examined whether loss of Nrf2 reversed the effects of propofol on cell proliferation, apoptosis, and invasion. Results showed that propofol alone and propofol plus sh-NC significantly promoted proliferation, stimulated invasion and inhibited apoptosis compared to parent cells. In contrast, propofol with ShRNA-1118 and ShRNA-2019 reversed these effects (Figure 4A to Figure 4D). Figure 4 Regulation of loss of Nrf2 for the effects of propofol on cell proliferation, apoptosis, and invasion. After transfected by different vectors, GBC-SD cells were incubated with propofol (20 μmol/L).