Addition of TIP decrease cellular FC levels in PC12 cells. (A) FC levels corrected for protein in PC12 cells. The concentration of NGF was 20 ng/ml. TIP consisted of 5 μg/ml transferrin, 10 μg/ml insulin, and 8 μM progesterone. The data were analyzed using a one-way ANOVA and are expressed as means ± SD (n = 3). (B) Time course of FC levels normalized to protein levels in PC12 cells treated without (black line) or with (gray line) TIP (5 μg/ml transferrin, 10 μg/ml insulin, and 8 μM progesterone). The data were analyzed using a Student’s t test and are expressed as means ± SD (n = 3). *p<0.05 and ***p<0.001 compared with and without TIP. (C) Measurement of FC levels in the presence insulin. In some experiments, transferrin and progesterone were also administrated. FC levels corrected for protein. The data were analyzed using a one-way ANOVA and are expressed as means ± SD (n = 3). ***p<0.001 compared with insulin (−). (D) Analysis of the concentration dependence of each factor of TIP. Concentration of 1 factor was changed in the presence of other 2 factors. FC levels corrected for protein. White bar: control cells cultured with serum without TIP. Gray bar: Cells cultured without serum with various concentration of TIP. The data were analyzed using a one-way ANOVA and are expressed as means ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.001 compared with 0 μg/ml transferrin, 0 μg/ml insulin, and 0 μM progesterone. #p<0.05, ##p<0.01 compared with 5 μg/ml transferrin, 10 μg/ml insulin, and 0 μM progesterone.