A) Effect of Different Concentrations of LY294002 on P53-Mediated Transcription

Supplementary Figures:

Figure SF1

A) Effect of different concentrations of LY294002 on p53-mediated transcription.

A549 cells were transfected with 1 µg of PG13-Luc. After 6 hrs of transfection, cells were treated with different concentrations of LY294002 as indicated and 1 hr later, adriamycin (1.0 µg/mL) was added. After 24 hrs of adriamycin addition, lysates were prepared and assayed for luciferase activity.

B) Effect of adriamycin induced DNA damage on PI3-kinase.

A549 cells were treated with LY294002 (50 µM) as indicated and 1 hr later, adriamycin (0.5 µg/mL) was added. At different time points after adriamycin addition (as indicated), lysates were prepared and assayed for Ser473 Akt and total Akt levels using PathScan ELISA kit (Cell signaling). The levels of Ser473 phospho AKT were normalized to total AKT in the same sample and also to the phosphorylation levels in the cells treated with only the vehicle (DMSO) to get the final values.

C) A549 cells were treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. After 24 hrs of adriamycin addition, total lysates were prepared and subjected to Western blot analysis for Ser166 phospho Mdm2, total Mdm2 and cyclophilin B (internal control) proteins using specific antibodies.

Figure SF2

p53 activation upon adriamycin induced DNA damage.

A549 cells were treated with adriamycin (0.5 µg/mL) and at indicated time points after adriamycin addition, total lysates were prepared and subjected to western blot analysis for Ser15 phospho p53, Ser20 phospho p53, Ser46 phospho p53, Ser392 phospho p53, Lys382Ac p53, total p53 and tubulin (internal control) proteins using specific antibodies.

Figure SF3

A549 cells were either untreated or treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. After 48 hrs of adriamycin addition, the cells were grown for 3 more days in drug free medium and photographed with a light microscope. Note the presence of viable cells in panels corresponding to LY294002 + adriamycin-high (0.75, 1.0 and 2.0 µg/mL; red panels) but not in LY294002 + adriamycin-low conditions (0.1, 0.25 and 0.4 µg/mL; blue panels).

Figure SF4

A, B) A549 cells were either untreated or treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. The adriamycin and LY294002 treatment was renewed every 48 hours and cells were photographed with a light microscope after 7 days (A) and 10 days (B) of first adriamycin addition.

Figure SF5

A) A549 cells were either untreated or treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. After 24 hrs of adriamycin addition, the cells were harvested and subjected to flow cytometry analysis. The proportion of cells in sub-G1 phase is shown in each panel.

B) A549 cells were either untreated or treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. After 48 hrs of adriamycin addition, the cells were harvested and subjected to flow cytometry analysis. The proportion of cells in sub-G1 phase is shown in each panel.

Figure SF6

A) LY294002 inhibits cisplatin induced p53 activation.

A549 cells were transfected with 1 µg of PG13-Luc and after 6 hrs of transfection, cells were treated with LY294002 (10 µM) and 1 hr later, cisplatin (indicated amounts) was added. After 24 hrs of cisplatin addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times and a representative experiment result is shown.

B) LY294002 inhibits etoposide induced p53 activation.

A549 cells were transfected with 1 µg of PG13-Luc and after 6 hrs of transfection, cells were treated with LY294002 (10 µM) and 1 hr later, etoposide (indicated amounts) was added. After 24 hrs of etoposide addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times and a representative experiment result is shown.

C) Effect of LY294002 on cisplatin induced cytotoxicity.

A549 cells were treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of cisplatin were added. After 48 hrs of cisplatin addition, proportion of live cells was quantified by MTT assay.

D) The chemosensitivity index (the ratio of cell viability of upon treatment with cisplatin alone and upon treatment with LY294002 and cisplatin) from C was calculated and shown in log scale. Please note that chemosensitivity index is high and low in cisplatin -low and –high conditions respectively.

Figure SF7

A) Hela cells were treated with LY294002 (50 µM), and 1 hr later, indicated concentrations of adriamycin were added. After 48 hrs of adriamycin addition, proportion of live cells was quantified by MTT assay.

B) The chemosensitivity index (the ratio of cell viability of upon treatment with adriamycin alone and upon treatment with LY294002 and adriamycin) from A was calculated and shown.

C) A549 cells were transfected with 1 µg of PG13-Luc. After 6 hrs of transfection, cells were treated with LY294002 (50 µM) and 1 hr later, indicated concentrations of adriamycin were added. After 24 hrs of adriamycin addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times and a representative experiment result is shown.

D) A549 cells were transfected with 1 µg of PG13-Luc and 2 µg of DNPI3K construct as indicated. After 6 hrs of transfection, cells were treated with LY294002 (50 µM) and 1 hr later, indicated concentrations of adriamycin were added. After 24 hrs of adriamycin addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times and a representative experiment result is shown.

Figure SF8

Effect of LY294002 treatment on the drug retention in treated cells

A549 cells were treated with DMSO or LY294002 and one hour later the indicated concentrations of adriamycin were added.24 hours after adriamycin addition, the cell were harvested by trypsinization and subjected to laser flow cytometry at an excitation wavelength of 480nm.

A) An overlay of the peaks corresponding to DMSO and LY294002 treated cells.

B) An overlay of peaks corresponding to DMSO, 0.25, 0.5 and 1 µg/ml Adriamycin to show a concentration dependent increase in the fluorescence.

C) An overlay of 0.25 µg/ml adriamycin treated sample with and without LY294002 pre-treatment.

D) An overlay of 0.5 µg/ml adriamycin treated sample with and without LY294002 pre-treatment.

E) An overlay of 1 µg/ml adriamycin treated sample with and without LY294002 pre-treatment.

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