Supplementary Data

Table 1. IC50 values (M) of gefitinib (A) and erlotinib (B) toward wild type and L858R EGFR. After

starvation, cells were preincubated with different concentrations of gefitinib or erlitinib for 30 minutes and then

stimulated with 100 ng/ml EGF for 2 minutes. Cell lysates were run on SDS-PAGE and receptor

phosphorylation at different tyrosine residues were detected by western blotting with specific phospho-EGFR

antibodies. In order to determine the concentration of kinase inhibitor (gefitinib or erlotinib) that resulted in 50% EGFR activity (IC50), the data were fitted to the following equation. Percent activity = (activitymax*IC50) / (IC50 + [kinase inhibitor]) + constant.

A.

B.

tyrosine residues wild type L858R

845 0.083 0.029 0.013 ± 0.002

992 0.020 0.002 0.018 0.003

1045 0.019 0.002 0.008 0.001

1068 0.074 0.022 0.054 0.015

1148 0.071 0.013 0.016 0.001

1173 0.037 0.006 0.010 0.002

tyrosine residues wild type L858R

845 0.150 0.046 0.005 0.001

992 0.015 0.002 0.010 0.001

1045 0.015 0.001 0.003 0.0002

1068 0.059 0.018 0.035 0.004

1148 0.034 0.003 0.018 0.004

1173 0.025 0.003 0.006 0.0006

Table 2. IC50 (M) of gefitinib (A) and erlotinib (B) toward downstream signaling proteins in 32D cells. After

starvation, cells were preincubated with different concentrations of gefitinib for 30 minutes and then stimulated

with 100 ng/ml EGF for 2 minutes. Cell lysates were run on SDS-PAGE and various EGFR signaling proteins

were detected by western blotting with total and phospho-antibodies. In order to determine the concentration of kinase inhibitor (gefitinib or erlotinib) that resulted in 50% EGFR activity (IC50), the data were fitted to the following equation. Percent activity = (activitymax*IC50) / (IC50 + [kinase inhibitor]) + constant.

A.

**shows no inhibition up to indicated concentrations of gefitinib

32D wild type 32D L858R Fold-difference

pCbl 0.05 0.007 0.0015 0.0002 33

pERK 0.84 0.24 0.019 0.009 44

pAKT 0.47 0.16 0.023 0.011 20

pPLC- 0.78 0.05 0.0048 0.0010 163

pSTAT3 0.0061 0.0009

0.00087 0.00038 7

pSrc 0.042 0.011 > 0.5** <0.1

pPI3K 0.46 0.10 > 0.5** <1

pSTAT5 0.64 0.54 0.0024 0.0011 267

B.

32D wild type 32D L858R Fold-difference

pCbl 3.74 0.36 0.013 0.0033 288

pERK 1.08 0.25 0.012 0.006 90

pAKT 0.92 0.22 0.028 0.013 33

pPLC- 0.13 0.02 0.008 0.002 16

pSTAT3 0.017 0.003 0.025 0.009 1

pSrc 4.12 5.58 > 0.5** <8

pPI3K 0.16 0.13 > 0.5** <0.3

pSTAT5 0.068 0.033 0.0038 0.0005 18

**shows no inhibition up to indicated

concentrations of erlotinib

Table 3. IC50 (M) of

gefitinib (A) and

erlotinib (B) toward

downstr

eam signaling proteins in cancer cell lines. After starvation, cells were preincubated with different

concentrations of gefitinib for 30 minutes and then stimulated with 100 ng/ml EGF for 2 minutes. Cell lysates

were run on SDS-PAGE and various EGFR signaling proteins were detected by western blotting with total and

phospho-antibodies. In order to determine the concentration of kinase inhibitor (gefitinib or erlotinib) that resulted in 50% EGFR activity (IC50), the data were fitted to the following equation. Percent activity = (activitymax*IC50) / (IC50 + [kinase inhibitor]) + constant.

A.

A431 H3255 Fold-difference

pCbl 0.016 0.0052 0.00016 0.00003 100

pERK 0.041 0.031 0.0015 0.0018 27

pAKT 0.10 0.077 0.0037 0.00003 27

pPLC- 0.046 0.014 0.0049 0.0052  9

pSTAT3 0.028 0.0087 0.0052 0.010 5

pSrc > 1** > 0.04**

pSTAT5 0.035 0.030 0.0080 0.017  4

**shows no inhibition up to indicated concentrations of gefitinib

A431 H3255 Fold-difference

pCbl 0.043 0.030 0.0012 0.00079 36

pERK 0.0045 0.0037 0.00032 0.00011 14

pAKT 0.10 0.16 0.0017 0.001  59

pPLC- 0.071 ± 0.003 0.0021 0.00088  34

pSTAT3 0.21 0.049 > 0.04**  < 5

pSrc > 1.0** > 0.04**

pSTAT5  0.021 0.0075 0.000052±0.00001  404

B.

**shows no inhibition up to indicated concentrations of erlotinib

Figure 1. (A) Effect of gefitinib on activation of EGFR signaling proteins in 32D cells. (B) Effect of erlotinib on

activation of EGFR signaling proteins in 32D cells. After serum starvation, cells were preincubated with the

indicated concentrations of gefitinib for 30 minutes and then stimulated with 100 ng/ml EGF for 2 minutes. Cell

lysates were run on SDS-PAGE and various EGFR signaling proteins were detected by western blotting with

total and phospho-antibodies.

(A)

pAKT

AKT

pERK

ERK

pPLC-

PLC-

pSTAT3

STAT3

gefitinib (M)pSrc

Src

wild type L834R

pCbl

pPI3K

Cbl

gefitinib (M)

wild type L834R

PI3K

pSTAT5

STAT5

(B)

erlotinib (M)

pAKT

AKT

pERK

ERK

pPLC-

PLC-

pSTAT3

STAT3

pSrc

Src

wild type L834R

pCbl

pPI3K

Cbl

erlotinib (M)

wild type L834R

PI3K

pSTAT5

STAT5

Figure 2. (A) Effect of gefitinib on activation of EGFR signaling proteins in cancer cells. (B) Effect of erlotinib

on activation of EGFR signaling proteins in cancer cells. After serum starvation, cells were preincubated with

the indicated concentrations of gefitinib for 30 minutes and then stimulated with 100 ng/ml EGF for 2 minutes.

Cell lysates were run on SDS-PAGE and various EGFR signaling proteins were detected by western blotting

with total and phospho-antibodies.

(A)

pAKT

AKT

pERK

ERK

pPLC-

PLC-

pSTAT3

STAT3

gefitinib (M)pSrc

Src

A431 H3255

pCbl

Cbl

A431 H3255

pSTAT5

STAT5

gefitinib (M)

(B)

erlotinib (M)

pAKT

AKT

pERK

ERK

pPLC-

PLC-

pSTAT3

STAT3

pSrc

Src

pCbl

Cbl

erlotinib (M)

pSTAT5

STAT5

A431 H3255 A431 H3255