Figure 2

Rapamycin induces key features of dormancy. Evaluation of cellular properties after treatment of KG1a cells with 100 nM rapamycin for 48 hours. (A) RNA content: Flow cytometric dotplots indicate the percentage of cells which are low in RNA (Pyronin Y) as well as low in DNA (7-AAD) and summary chart. In the summary chart, the RNA content of KG1a cells treated for 48 hours with etoposide, which induces predominant G2/M arrest, are used as negative control. (B) The total RNA content of lysed cells, measured by spectrophotometry. (C) Flow cytometric analysis of forward scatter as an indicator of cell size: representative histogram: dark-filled histogram = untreated cells, unfilled histogram = rapamycin-treated cells and summary graph (instrument and voltage-dependent units). (D) Reduction of 2,3-bis(2-methoxy-4-nitro5-sulfophenyl)-5-{(phenylamino)carbonyl}-2H-tetrazolium hydroxide to formazan: absorbance ratio (adjusted for cell count) after 48 hours as a measure of mitochondrial metabolism. (E) Flow cytometric dichlorofluorescein diacetate as a measure of reactive oxygen species: example, dark-filled histogram = untreated cells, unfilled histogram = rapamycin-treated cells, pale grey histogram = baseline; summary graph. In each experiment datapoints illustrate mean and standard deviation of at least 3 independent assays. All P values are for comparisons between untreated and rapamycin-treated cells.