Research Paper Volume 7, Issue 10 pp 869—881

Figure 5. ^mtROS are involved in the induction of mitonuclear stress response and uncoupling.

(A) Cytofluorimetric detection of ^mtROS after staining with MitoSOX. (B) Protein levels of Drp1, SOD2 and UCP1 analyzed by Western blot. (C) Mitochondrial fragmentation detected through confocal microscopy in cells transfected with mitochondrial Ds-Red fluorescent protein and treated or not with NAC. (D, E) OxPHOS gene expression ratio evaluated by calculating the ratio between ^mtDNA-encoded (MTCO1 and ATP6) and ⁿDNA-encoded (SDHA and Cox4b) mitochondrial mRNAs after RT-qPCR in cells treated or not with NAC. (F) OxPHOS protein ratio (right panel) evaluated by calculating the ratio between ^mtDNA-encoded (MTCO1) and ⁿDNA-encoded (NDUFB8) mitochondrial proteins (left panel) after Western blot followed by densitometric analysis. (G, H) mRNA levels of UCP1 and ClpP measured through RT-qPCR in cells treated or not with NAC. (I) Cytofluorimetric detection of mitochondrial amount and membrane potential through MitoTracker Green (MTG) and MitoTracker Red CMX-ROS (MTR) staining, respectively. Polarized mitochondria determined by calculating Red-to-Green ratio (MTR/MTG). TOMM20 and vDAC staining served as loading control. Bar graphs are expressed as mean ±S.D. (n=4; *p<0.05; **p<0.01 vs CM; °p<0.05). NR: nutrient restriction; CM: complete medium.