Figure 1. ACC1 levels, expression and lipid synthesis decrease immediately after the exposure to doxorubicin.

Early passage human fibroblasts were exposed for twenty-four hours to either doxorubicin 0.2 μM (Doxo) or the vehicle DMSO, (Control, Ctl) in complete media. All measurements were done at the end of the treatment, unless otherwise specified. (A) ACC1 levels, ATM phosphorylation (p-ATM), p53 phosphorylation (p-p53), p53, p21 and pRb phosphorylation (p-pRB) were assessed by Western blot at different times during the incubation, tubulin was used as loading control. The graph on the right shows the data obtained after quantifying the blots and normalizing ACC1 and p-ATM levels to tubulin. Control cells were incubated with DMSO for 24 hours. (B) γ-H2AX foci were assessed by immunofluorescence, original magnification X400. The percentage of cells with nuclear foci is shown below the picture. (C) Cell proliferation was measured by immunofluorescence, as BrdU incorporation to nuclear DNA, original magnification X400. The percentage of cells with positive nuclear staining is shown below the picture. (D) SA-β-Gal staining was assessed by light microscopy, original magnification X40, 15 days after exposure to doxorubicin. The percentage of positive cells is shown below the picture. (E) Twenty-four hours after the exposure to the drug ACC1 and tubulin levels were determined in independent Western blots and protein levels of ACC1 were normalized using tubulin as loading control. (F) ACC1 mRNA levels. (G) Lipid synthesis was determined by ¹⁴C-acetate incorporation. Results are expressed as the mean ± SEM (n≥3, *P<0.05, **P<0.005, ***P<0.0005).