Supplementary Information

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1 Supplementary Information EGFRL858R mutant enhances lung adenocarcinoma cell invasive aility and promotes malignant pleural effusion formation through activation of the CXCL12CXCR4 pathway MengFeng Tsai 1*, TzuHua Chang 2*, ShangGin Wu 3, HsiaoYin Yang 2, Yi Chiung Hsu 5, PanChyr Yang 2,4 & JinYuan Shih 2,4 Affiliation of authors: 1Department of Molecular Biotechnology, College of Biotechnology and Bioresources, Dayeh University, Changhua 51591, Taiwan, 2Department of Internal Medicine, National Taiwan University Hospital, and College of Medicine, National Taiwan University, Taipei 12, Taiwan, 3Department of Internal Medicine, National Taiwan University Hospital, YunLin Branch, Yunlin 6441, Taiwan, 4 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei 12, Taiwan, 5Institute of Statistical Science, Academia Sinica, Taipei 11529, Taiwan. *These two authors contriuted equally to this work. MengFeng Tsai & TzuHua Chang Corresponding author: JinYuan Shih, MD, PhD, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, 7 ChungShan South Road, Taipei 12, Taiwan. Telephone: ext Fax: address: jyshih@ntu.edu.tw Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure S3 Supplementary Figure S4 Supplementary Tale S1

2 No. of cells No. of cells a 3 25 ANo. of cells H1299EGFRWT H1299EGFR_L85R B days H1299EGFRWTsiScramle H1299EGFRWTsiCXCR4 H1299EGFRL858RsiScramle H1299EGFRL858RsiCXCR days Figure S1. Cancer cell proliferation rates assessed y thiazolyl lue tetrazolium romide (MTT) assay. (a) The cell proliferation rates of H1299 EGFRWT and H1299 EGFRL858R lung cancer cells. The proliferation rates of these two cells have no difference. () CXCR4 in H1299EGFRWT and H1299EGFRL858R cells was knocked down using a CXCR4 specific sirna. Cells were harvested 24 hours after transfection, and than the cell proliferation rates of H1299EGFRWTsiScramle, H1299EGFRWTsiCXCR4, H1299 EGFRL858RsiScramle and H1299 EGFRL858RsiCXCR4 lung cancer cell were analyzed. siscramle, cells transfected with scramled control sirna; sicxcr4, cells transfected with CXCR4specific sirna. The proliferation rates of these four cells have no difference.

3 a H1299 EGFRWT H1299 EGFRL858R EGFR 18 kda EGFR 18 kda βactin 42 kda βactin 42 kda c EGFR 18kDa βactin 42kDa Figure S2 EGFRL858R expression in lung cancer cell. (a) Overexpression of EGFRWT and EGFRL858R in lung adenocarcinoma H1299 cells was evaluated y Western lotting. () Small interfering RNA (sirna) specifically targeting EGFRL858R knocked down EGFRL858R protein expression in H1299EGFRL858R cells. (c) Overexpression of EGFRWT and EGFR L858R in lung adenocarcinoma CL1 cells was evaluated y Western lotting. This is a full length image of the cropped lot presented in the Figure 1a, c and e.

4 a c d Figure S3 The effective inhiition concentration of pharmacological inhiitors (EGFR inhiitor AG1478, ERK inhiitor U126, PI3KAKT inhiitor LY2942 and JAKSTAT3 inhiitor AG49) were performed in H1299EGFRL858R cells and then stimulated with 4ng/ml EGF. The phosphorylation status of EGFR, AKT, ERK1/2 and STAT3 (the main downstream effectors of EGF signaling pathways) was determined y Western lotting. (a) EGFR and phosphoegfr levels were evaluated in H1299EGFRL858R cells pretreated with different concentrations of the EGFR inhiitor, AG1478. () ERK and phosphoerk levels were evaluated in H1299EGFR L858R cells pretreated with different concentrations of the ERK inhiitor, U126. (c) AKT and phosphoakt levels were evaluated in H1299EGFRL858R cells pretreated with different concentrations of the PI3KAKT inhiitor, LY2942. (d) STAT3 and phosphostat3 levels were evaluated in H1299EGFRL858R cells pretreated with different concentrations of the JAK STAT3 inhiitor, AG49.

5 a H1299 EGFRL858R + + AG1478 (1 μm) + + EGF (4ng/ml) H1299 EGFRL858R + + U126 (1 μm) + + EGF (4ng/ml) pegfr (18kDa) pegfr (18kDa) EGFR (18kDa) perk EGFR (18kDa) perk ERK ERK pakt (6kDa) pakt (6kDa) AKT (6kDa) AKT (6kDa) pstat3 (86kDa) pstat3 (86kDa) STAT3 (86kDa) STAT3 (86kDa) βactin βactin Figure S4. (a) The effective inhiition concentration of EGFR inhiitor AG1478 (1um) were used and the phosphorylated and total protein of EGFR, ERK, AKT, and STAT3 were detected y Western lotting in H1299EGFRL858R cells with or without EFG stimulation. () The effective inhiition concentration of ERK inhiitor U126 (1uM) were used and the phosphorylated and total protein of EGFR, ERK, AKT, and STAT3 were detected y Western lotting in H1299EGFRL858R cells with or without EFG stimulation.the results showed that EGFR inhiitor AG1478 were significantly inhiition the phosphorylatedegfr, phosphorylatederk, phosphorylatedakt, ut not phosphorylatedstat3. Using ERK inhiitor U126 treated the H1299EGFRL858R cells, indicated that only phosphorylatederk was aolished This is a full length image of the cropped lot presented in the Figure 4,c.

6 Tale S1: Clinical characteristics of the 12 lung adenocarcinoma patients with malignant pleural effusion (MPE) Variale L848R WT Pvalue Total No. 6 6 Sex Male Female 2 2 Age Median * (Range) ( ) ( ) Smoking Never Current/Former 3 3 T N *By MannWhitney U test To confirm the association etween CXCR4 and the EGFRL858R mutation, we performed an EGFR mutation analysis of cancer cells in MPEs from 12 lung adenocarcinomas and analyzed the surface expression of CXCR4. The clinical characteristics of the 12 patients, shown in supplementary Tale S1.