Figure 4. Impaired fatty acid synthesis, but not exogenous fatty acid depletion, arrests the cells at G₂/M. HeLa cells were incubated with 20 μg/ml of fatty acid synthase inhibitor (C75) or vehicle (DMSO) for 24 h. Cell extracts were assayed for FASN activity (A). Alternatively, the cells were pulsed with BrdU and the cell cycle distribution was assessed by FACS (B) and immunoblotting analysis of cyclins levels (C). To assess the importance of exogenous fatty acids for cell cycle progression, the cells were incubated with regular or charcoal-stripped FBS (CS-FBS) for 24 h and analyzed by FACS (D, inset). The sustained effect of exogenous lipid depletion on proliferation was estimated by counting of cells grown in CS-FBS for up to 72 h (D). *P < 0.0001 Chi-square and Fisher Exact Test (B).