Fig. 3

Analysis of mitochondrial capacity and signaling pathways of macrolide antibiotic-treated in vitro differentiated Th17 cells. a FACS analysis of mitochondrial cytochrome c oxidase depicted as mean fluorescent intensity (MFI) at day 4 of culture, data shown are representative of one out of two independent experiments, each performed in technical triplicates and depicted as mean ± S.E.M. b MFI of MitoTracker Deep Red™ at day 4 of Th17 culture, data shown are representative of one out of three independent experiments, each performed in technical duplicates and depicted as mean ± S.E.M. c Oxygen consumption rate of cells at day 3 of Th17 culture in Mito-stress test with subsequent injections of oligomycin, FCCP, rotenone, and antimycin A at the indicated time points. Data from one out of two experiments, each performed in technical triplicates and depicted as mean ± S.E.M. d Western blot of cell lysates to identify phosphorylated and unphosphorylated p70-S6-kinase at 24 h of culture, e at 45 and 60 min. Membranes were subsequently probed twice with antibodies to visualize phosphorylated and total protein as indicated with a sign plus as superscript 2. f Western blot to identify p90-S6-kinase 24 h after start of culture. g Quantification of p-p70-S6/p70-S6 and p-p90-S6/p90-S6 of three independent Western blots from independent cultures at 24 h normalized to the respective ratios in DMSO controls. All Western blots for indicated time points were run at least twice and verified initial findings. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. ns (not significantly different); *p < 0.05