Figure 2. Leucine-dependent growth occurs at low leucine concentrations and is modulated by BSA.

(A) Time course of cell growth in R(0), R(Leu), and R(BSA), +/− CSF1, as measured hourly. Measurements from every 4 hrs are shown. Data show mean and SEM obtained from 9 wells per time point (3 biological replicates, 3 wells per condition).

(B) Rates of cell growth in R(0), R(Leu), R(BSA), +/− CSF1 from the time courses in panel A, showing hourly measurements normalized to the cell counts for each condition at 30 hrs. Lines show linear regression analysis (n = 81 per condition).

(C) Slopes of the lines shown in panel B. The relative rates of cells in media with CSF1 were highly significant (One-way ANOVA, ****, p<0.0001).

(D) Rates of cell proliferation in R(0) (blue) and in R(0) supplemented with the indicated concentrations of leucine (green). Cell numbers per well were measured every hour and normalized to the cell numbers per well at 30 hrs (as in panel 1B). Each point indicates the mean and SEM of a slope determined from 81 measurements (9 time points from 30–38 hrs, 3 wells per time point, 3 biological replicates).

(E) Effects of BSA (10 mg_*ml⁻¹) on rates of cell proliferation in different concentrations of leucine. Each point indicates the mean and SEM of a slope determined from 81 measurements (9 time points from 30–38 hrs, 3 wells per time point, 3 biological replicates). BSA stimulated growth in low concentrations of leucine and reduced growth in high concentrations of leucine. The difference at each concentration of leucine with and without BSA was highly significant (Ordinary one-way ANOVA, ****, p<0.0001).

(F) Protein measurements taken at the end of the experiments shown in panel E, obtained from lysates of cells at 48 hrs. The difference with and without BSA was significant in some but not all concentrations of leucine (Student’s T-test of pairs at each leucine concentration; **, p<0.01, *, p<0.05, ns: not significant, n=9 wells per condition, 3 × 3 biological replicates).