Fig. 5

Analysis of RC assembly and activities after Tig/Tet treatment. Low and high metastatic CRC cells were treated with Tig/Tet for 24 h, and mitochondria were isolated for analyzing RC complex assembly by BN-PAGE and activities by in-gel assays as described in the Methods section. To ensure equal loading of samples, denaturing SDS-PAGE was performed in parallel, followed by immunoblotting with mitochondrial loading control VDAC. Results of Tig/Tet treatment in A low metastatic and B high metastatic cells are presented as a RC assembly showing Coomassie blue stained image and loading control VDAC blot; b-d relative fold change in band intensities of C-I b, C-V c and C-IV d as analyzed by densitometry. Band intensities of Coomassie-stained bands were normalized with the band intensity of corresponding VDAC values and are presented as fold change relative to non-treated control. (B-e) In addition, gel assay results of different RC complexes in high metastatic cells after treatment are shown. The values represent the fold change relative to the non-treated control. These experiments were performed 2–3 times, and the images are representative of one of the experiments with similar results. Abbreviations: RC, Respiratory complex; VDAC, Voltage-dependent anion channel; BN-PAGE, Blue native polyacrylamide gel electrophoresis