Hk2 deletion blunts aggressiveness of hGFAP-cre;SmoM2-driven medulloblastoma and extends survival. (A) Western blot comparing expression of Hk2 and Hk1 in mice with indicated genotype at P7 and P15. Expression of Hk2 protein in SmoM2 tumors with Hk2+/+ genotype exceeded the expression of Hk2 in wildtype mice at P7. Hk2 protein was absent in SmoM2 tumors with the Hk2fl/fl genotype. (B) Comparison of total Hk activity in tumors with the Hk2+/+ or Hk2fl/fl genotype. Two-tailed Student’s t test used for statistical comparison. (C) Kaplan–Meier curves demonstrate increased event-free survival in hGFAP-cre;SmoM2;Hk2fl/fl mice, compared with hGFAP-cre;SmoM2;Hk2+/+ mice (P < 0.00002, log-rank test). While 100% (24/24) of hGFAP-cre;SmoM2;Hk2+/+ mice developed symptomatic tumor requiring euthanasia by P20, 30% (4/13) of hGFAP-cre;SmoM2;Hk2fl/fl mice remained alive without symptoms at P100. (D) Western blot for GFP and Smoothened (Smo) demonstrates expression of SmoM2-YFP fusion protein (arrowhead) in hGFAP-cre;SmoM2 mice with either the Hkfl/fl or the Hk2+/+ genotype. The SmoM2 allele is detected in the Smoothened blot as a band (arrowhead) that is not present in wildtype P8 cerebella and is of higher molecular weight than the wildtype protein. (E) IHC for GFP (brown) demonstrates SmoM2-YFP protein in the neoplastic and differentiated regions of cerebella from hGFAP-cre;SmoM2 mice with either the Hkfl/+ genotype (upper panel) or the Hk2 fl/fl genotype (middle panel). A section of cerebella from a wildtype mouse, processed in parallel, demonstrated absence of staining with GFP antibody under identical conditions (lower panel). Nuclei are counterstained blue with hematoxylin. Scale bars = 50 μm.