Both Nova2 knockdown and dual sevoflurane exposure suppressed Netrin-1/Dcc protein expression. (A) QPCR revealed that Netrin-1 mRNA expression was decreased among 28 candidate genes in the Nova2 shRNA group (F = 0.76, P = 0.0015**, N = 3, Student’s t-test), while Dcc mRNA expression wasn’t change (F = 0.699, P = 0.0519, N = 3, Student’s t-test). (B) Western blot analysis demonstrated that Nova2 knockdown suppressed Dcc and Netrin-1 proteins expressions. (C) Quantification of the protein expression of Dcc relative to Actin (P = 0.0217*, N = 3, Student’s t-test). (D) Quantification of the protein expression of Netrin-1 relative to Actin (P = 0.0264*, N = 3, Student’s t-test). (E) Western blot analysis demonstrated that Nova2 OE upregulated Dcc and Netrin-1 protein expressions in the cortical tissues of offspring mice. (F) Quantification of the protein expression of Dcc relative to Actin (P = 0.0148*, N = 3, Student’s t-test). (G) Quantification of the protein expression of Netrin-1 relative to Actin (P = 0.0353*, N = 3, Student’s t-test). (H) Western blot analysis demonstrated that dual sevoflurane exposure also decreased Dcc and Netrin-1 proteins expressions. (I) Quantification of the protein expressions of Dcc (F = 1.118, P = 0.0044**, N = 3, Student’s t-test) and Netrin-1 (F = 0.386, P = 0.0052**, N = 3, Student’s t-test) relative to Actin. *P<0.05. **P<0.01, ***P<0.001.****P < 0.0001.