Fig 2: Nova2 regulates neuronal migration through modulating Netrin-1/Dcc activity in the developing mouse cerebral cortex. (A) Dual sevoflurane exposure significantly decreased neuronal migration cortex of offspring mice, and Nova2 overexpression can rescue the neuronal migration deficits induced by dual sevoflurane exposure. While Netrin-1/Dcc shRNA mitigated the Nova2 rescue phenomenon, which was similar to the neuronal migration deficits induced by dual sevoflurane exposure. (B) Quantification of GFP+ cells at different positions in different groups. Similar with dual sevoflurane exposure group, Netrin-1/Dcc knockdown mitigated the Nova2 rescue phenomenon, larger fractions of neurons in the IZ (F = 5.672, P = 0.0634, N = 3, one-way ANOVA) and the VZ/SVZ (F =14.68, P = 0.0126*, N = 3, one-way ANOVA), and a significantly smaller fraction of neurons in the UpCP (F = 35.79, P = 0.0024**, one-way ANOVA). *P<0.05. **P<0.01, ***P<0.001.****P < 0.0001.

Fig 3: 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.

Fig 4: (A) TRAP staining of raw264.7 and osteoclasts. (B) Cellular immunofluorescence of Netrin-1, Unc5B and DCC on osteoclasts. Arrow: the expression of Netrin-1 and Unc5B on the membrane of the osteoclasts.