Figure 4. SIRT1 Regulates HIF-1α Stabilization in Skeletal Muscle through Regulation of VHL Expression.

(A and B) Immunoblot for VHL and tubulin in gastrocnemius of WT and SIRT1 iKO mice (A) and WT and SIRT1-Tg mice (B).

(C and D) VHL mRNA in WT and SIRT1 iKO mice (C) and WT and SIRT1-Tg mice (D). Values normalized to WT mice (n = 5, *p < 0.05 versus WT).

(E) Immunoblot for VHL, HIF-1α, and tubulin in gastrocnemius of 6- and 22-month-old mice.

(F) VHL promoter activity in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 hr to induce SIRT1 excision (SIRT1 iKO). Luciferase values normalized to vehicle cells (n = 5).

(G) VHL promoter activity in primary myoblasts with adenovirus expressing SIRT1 or empty vector. Luciferase values normalized to empty vector cells (n = 5).

(H) Immunoblot for VHL and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with VHL or nontargeting shRNA.

(I) Representative immunoblot for HIF-1α in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with VHL or nontargeting shRNA and treated with OHT for 24 hr (SIRT1 iKO), after which SIRT1 was added back by adenoviral infection.

(J) Expression of mitochondrially encoded genes in primary myoblasts transduced with VHL or nontargeting shRNA and treated with adenovirus expressing SIRT1 or empty vector (n = 5, *p < 0.05 versus shNT empty; #p < 0.05 versus shNT SIRT1 OE). Mitochondrially encoded genes were ND1, Cytb, COX1, and ATP6.

Values are expressed as mean ± SEM. See also Figure S4.