Figure 3

Westernblot analysis of 4E-BP phosphorylation in parental and cisplatin resistant small cell lung cancer cell lines. A: SCLC1 (parental line) was treated with 0.1 ug/ml of cisplatin alone, CCI-779 alone or in combination. Lane1: control (untreated cells) which showed high amount of phosphorylated 4E-BP. Lane 2: SCLC1 treated with 0.1 ug/ml of cisplatin which showed a decrease in the amount of phosphorylated 4E-BP, lane 3&4: SCLC1 treated with 150 ng/ml of CCI-779 alone and CCI-779 with cisplatin 0.1 ug/ml respectively. Both showed comparable amount of phosphorylated 4E-BP and are less than control cells and or cells treated with cisplatin alone. B: 4E-BP phosphorylation in SR-2 (cisplatin resistant cell line derived from SCLC1) after treatment with cisplatin with and without CCI-779. Lane 1: Control untreated cells which showed an abundant amount of phospho-4E-BP, lane 2, 3, and 4: SR-2 treated with 5,10 and 0.1 ug/ml of cisplatin. The amount of phospho-4E-BP are not affected by cisplatin at low dose, but slightly decreased at high dose. Lane 5,6 SR-2 treated with CCI-779 at 150 ng and 300 ng/ml respectively. Predominantly unphosphorylated 4E-BP is seen, Lane 7&8: SR-2 treated with 150 ng/ml of CCI-779 combined with cisplatin 5 ug and 10 ug/ml respectively. Small amounts of phosphorylated 4E-BP are seen and much less than those with cisplatin alone (lane 2,3&4). C:4E-BP phosphorylation in SCLCB(parental line). Lane 1: Control untreated cells which showed an abundant amount of phospho-4E-BP, lane 2: SCLCB treated with 0.1 ug/ml of cisplatin showed a decrease amount of phospho-4E-BP. lane 3 & 4 SCLCB treated with 150 ng/ml of CCI-779 alone and CCI-779 150 ng/ml with 0.1 ug/ml of cisplatin respectively. Both showed comparable amount of phospho-4E-BP and is slightly less than cisplatin alone. D: 4E-BP phosphorylation in SCLCBC (cisplatin resistant cells). Lane 1,2,3: SCLCBC treated with CCI-779 150 ng/ml combined with cisplatin at 1,5 and 10 ug/ml of cisplatin respectively. The amount of phospho-4E-BP is minimum and predominantly unphosphorylated form, lane 4&5 SCLCBC treated with CCI-779 at 150 ng and 300 ng/ml, only unphosphorylated 4E-BP predominate. Lane 6,7,8 SCLCBC treated with 10,5 and 1 ug/ml of cisplatin respectively. The amount of phospho4E-BP are similar to the control cells. Lane 9. Control untreated cells. Actin is used as control for protein loading for all cell lines