Figure 6.
Mitochondrial respiration controls proliferation of lung cancer stem-like cells. (A) Western blotting of primary KRas;rank+/+ and KRas;rankfl/fl pneumocytes to determine activation of the indicated signaling pathway in response to 1 µg/mL RANKL stimulation. Activation was determined at the indicated time points using phospho-specific antibodies. The respective total proteins are shown to control for protein expression. β-Actin is shown as a loading control. (B) Bioenergetics profiling of purified primary pneumocytes from KRas;rank+/+ mice. Mutant KRas was induced following AdCre infections, and cells were then left untreated (control) or treated with 1 µg/mL RANKL in the presence or absence of inhibitors to block NF-κB (iNF-κB), AKT (iAKT), P38 (iP38), and transcription (actinomycin D). In all experiments, cells were pretreated with the inhibitors. A minimum of five replicates was analyzed for each condition, and pneumocytes purified from two different mice were used independently. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, unpaired two-sided t-test. (C) Representative images for BrdU staining of tumor spheroids derived from KRas;rank+/+ primary lung tumor cells, which received no treatment or were treated with 1 µg/mL RANKL alone, oligomycin alone (low dose [0.05 µg/mL] or high dose [0.5 µg/mL]), and RANKL plus different oligomycin concentrations. Five-thousand primary tumor cells were seeded. BrdU labeling (10 µM/mL) was performed for 2 h. Experiments were performed with six replicates for each condition and repeated with three different KRas;rank+/+ mice. Sections were counterstained with DAPI. (D,E) Quantifications (mean ± SEM) of tumor spheroid numbers and BrdU+ cells within tumor spheroids from C. (***) P < 0.001, unpaired two-sided t-test. Bars, 50 µm.