Figure 6: Neonatal HX promotes interactions between Sirt1 and specific components of the Cdk2 pathway.

Representative western blots of Cdks/Sirt1 (a) and Rb/Sirt1 (d) complexes in NX and HX white matter (P18). Increased pSirt1 Ser47/Cdk2, Sirt1/Cyc E (b) and Rb/Sirt1 (e), and lower Rb/acetyl lysine (e) expression after HX. Histograms show mean±s.e.m.(n=3 brains per condition). (c) IP analysis in NX and HX cells transfected with Cdk2 siRNA shows less phosphorylated Sirt1 (n=3 brains per condition). (f) Representative western blot of FACS-purified NG2⁺ cells from CNP-EGFP mice (P18). (g) Western blot showing HX enhanced formation of Rb/Cdk2 and Rb/Sirt1, and reduced Rb/acetyl lysine and Rb/E2F1 (n=9 brains for each condition). (h) Representative western blot showing sirtinol treatment enhances Sirt1-mediated Rb acetylation in NX. In HX, deacetylation is reduced and Rb/Cdk2 expression. (i) Images of NX and HX cells stained with anti-Sirt1 and anti-Rb antibodies and DAPI. Scale bar, 50 μm. (j) Graph represents higher percentage of Sirt1⁺Rb⁺ cells in cytoplasm after HX. Histograms show mean±s.e.m. (three NX and four HX brains).