Fig. 4

Mitochondrial mass and activity are impaired by AMPK catalytic subunits knockout in human GB cell lines

(A) LNT-229 and G55T2 wildtype (wt) and AMPK catalytic subunits double knockout (DKO) cells were analyzed for the mRNA expression of mtDNA D-loop by qPCR. 18 S and SDHA were used for normalization (n = 3, mean ± SD). (B) mRNA expression of mitochondrial encoded as well as mitochondrial associated genes (ATP5G1, MT-CYB, MT-ND1 and MT-CO1) of G55T2 wildtype and AMPK DKO cells was determined by qPCR. 18 S and SDHA were used as housekeeping genes for normalization (n = 3, mean ± SD). (C) G55T2 wildtype and AMPK DKO cells were incubated in serum-free DMEM for 24 h. Cells were stained with 100 nM MitoTracker Green or 100 nM MitoTracker Red for 20 min and analyzed by flow cytometry. Mean fluorescence intensities are shown (n = 3, mean ± SD, *p < 0.05, **p < 0.01, Student’s t-test). (D) LNT-229 wildtype and AMPK DKO cells were treated as described in (C). Bright-field (upper panel) and fluorescence microscopy (RFP channel: middle panel, GFP channel: lower panel) were used for analysis (48x magnification). Representative images are shown