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.

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

A.

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

B.

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.

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

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.

**shows no inhibition up to indicated concentrations of erlotinib

Table 3. IC50 (M) of gefitinib (A) and erlotinib (B) toward downstream 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)

(B)

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)

(B)

pAKTAKTpERKERKpPLC-PLC-pSTAT3STAT3

gefitinib(M)

pSrcSrcA431H3255pCblCblA431H3255pSTAT5STAT5

gefitinib(M)

erlotinib(M)

pAKTAKTpERKERKpPLC-PLC-pSTAT3STAT3pSrcSrcpCblCbl

erlotinib (M)

pSTAT5STAT5A431H3255A431H3255

pAKTAKTpERKERKpPLC-PLC-pSTAT3STAT3

gefitinib(M)

pSrcSrcwild typeL834RpCblpPI3KCbl

gefitinib(M)

wild typeL834RPI3KpSTAT5STAT5

erlotinib(M)

pAKTAKTpERKERKpPLC-PLC-pSTAT3STAT3pSrcSrcwild typeL834RpCblpPI3KCbl

erlotinib (M)

wild typeL834RPI3KpSTAT5STAT5