Figure 4.

mTORC2 regulates mammalian cell size and cell cycle progression. Logarithmically growing IMR-90 fibroblasts were transfected with short-interfering RNAs (siRNAs) targeting human raptor and rictor, respectively. Cells treated with non-targeting siRNA were analysed in parallel and served as a negative control (control). Forty-eight hours after transfection, cells were replated at low density and were grown for another 20 h. (A) Cells as described above were lysed and examined for the expression level of raptor, rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K. α-Tubulin was co-analysed as an additional loading control. (B) Cells derived from the same pool of cells described in (A) were cytofluorometrically analysed for DNA distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. Apart from the quantification of DNA distribution, cells were cytofluorometrically examined for overall cell size via FSC analyses (C) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA content (D).