Figure 3: Knockdown of α-SNAP elevates mitochondrial respiration and stimulates mitochondrial biogenesis.

(a) OCR (pmol/min) in HEK293T cells measured by a Seahorse Analyser. Lentiviral shRNA infected cells are indicated and double knockdown was performed as Fig. 2d. Basal measurements were normalized for protein concentrations and the mean±s.e.m. from four independent experiments are plotted. Student’s t-test was used to calculate significance. (b) Seahorse continuous measurement of OCR. (c) HEK293T cells were infected with lentiviral shRNA targeting GFP (control, CON) or α-SNAP (SNAP sh1 and sh2). Mitochondrial proteins were detected by immunoblotting: CV-α, ATP synthase subunit alpha; CIII-core2, Complex III subunit Core 2; COXIV, cytochrome c oxidase subunit IV. (d,e) Citrate synthase activity as a measure of mitochondrial content was assayed and normalized relative to controls. Data represent mean±s.e.m. (n=4) and P-values from Student’s t-test of significance are indicated. (d) Cells prepared as (c). (e) α-SNAP and LKB1 were knocked down as described in Fig. 2d.