SUPPLEMENTARYFIGURE LEGENDS
Figure S1: Nuclear localization of EGFR was enhanced by etoposide or doxorubicinin Hs578T breast cancer cells. Hs578Tbreastcancercells weretreatedwith etoposide(20μM)(A) or doxorubicin (1 μM) (B) for the indicated times,andnuclearfractionswereisolatedandimmunoblottedtodeterminethe total levels of EGFR and DNA-PKcs. Tubulin, lamin B1 and TIM23 were measured as markers for cytosol, nuclei and mitochondria, respectively. Expression of EGFR was quantified by densitometry, normalized to Lamin B1 as a nuclear fraction marker. Data are mean ±SD from three experiments. * P<0.05 compared to corresponding control value.
Figure S2:IGFBP-3 but not other IGF-related proteins interact with EGFR.Proximity ligation assays in MDA-MB 468 cell between EGFR and IGFBP-3, EGFR and IGF-II, or EGFR and IGFBP-1 were performed as described in Material and Methods. Each red spot represents a single interaction and DNA was stained with DAPI.
Figure S3:Effect of IGFBP-3 downregulation on the interaction between EGFR and DNA-PKcs in MDA-MB-468 cells. Cells were transfected with control siRNA or IGFBP-3 siRNA #1 as described under Materials and Methods. Cells were changed to serum-free medium 24 h after transfection and incubated for a further 24 h before being treated with or without etoposide (20 µM) for 4 h. Proximity ligation assay between EGFR and DNA-PKcs was performed (A) and the intracellular distribution of fluorescence intensity was quantified(B). The proportion of nuclear interactions as a percentage of total (%N) is indicated under each bar. Data are mean ± SD from three experiments. * P<0.05 compared to corresponding control value.
Figure S4: IGFBP-3 downregulation enhances Hs578T cell survival after etoposide or doxorubicin treatment. Cells were transfected with control or anti-IGFBP-3 siRNA and 500 cells/well were plated into 6-well plates for 24 h. The cells were then treated with either etoposide (5 µM) or doxorubicin (0.1 µM) for 4 h, and incubated in growth mediumunder humidified 5% CO2 at 37°C for 8-10 days to allow colony formation. Colonies having >50 cells were counted. (A) Representative wells showing colonies stained with crystal violet. (B) Mean values ± SD (3 experiments).* P<0.01 compared to corresponding control siRNA-treated cells.
Figure S5: Effect of doxorubicin on the distribution of EGFR and DNA-PKcs in lipid raft components. MDA-MB-468 breastcancercells weretreatedwith(A) doxorubicin(Dox, 1μM) or gefitinib (Gef, 10 µM) alone,or(B) doxorubicin combined with gefitinib for 6 or 24 h as indicated, and lipid raft membrane components were extracted in 1% Triton X-100 at 4 °C and fractionatedby centrifugation in a linear 5–30% sucrose gradient as described in Materials and Methods. Thepeak lipid raft-containing fractions (#4, arrowed, indicated by the presence of caveolin-1) were analyzed by Western blotting. DNA-PKcs and EGFR were quantified by densitometry, and values normalized to caveolin-1. Data are mean ± SD from three experiments. * P<0.05 compared to corresponding control value.
1