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Additional Supporting Information may be found in the online version of this article.

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IJC_25472_sm_suppinfofig1.tif798KSupplementary Figure 1. Overall survival rates of 29 breast cancer patients who could be followed-up among 52 breast cancer patients in relation to the tumor expression statuses of EGFR ligands. High expressions: AR (A), blue; TGFα (B), pink; EGF (C), green. Low expression of each EGFR ligand: black line. High or low expression of the EGFR ligands was defined as described in the Materials and Methods. The differences between the curves were estimated by the log-rank test, and a value of P<0.05 was considered statistically significant.
IJC_25472_sm_suppinfofig2.tif1624KSupplementary Figure 2. Expressions of EGFR, HER2, HER3, HER4, ER and PR in breast cancer cell lines. (A) The protein levels of EGFR, HER2, HER3, HER4 and?β-actin were evaluated by immunoblotting. (B) The mRNA levels of ER, PR and GAPDH were analyzed by RT-PCR.
IJC_25472_sm_suppinfofig3.tif1542KSupplementary Figure 3. Expressions of EGFR ligands in breast cancer cell lines. (A) The mRNA indexes of the EGFR ligands were evaluated by real-time PCR. The data represent the means ± SD of six experiments. *P<0.05, versus the RNA expression indexes of the other EGFR ligands. The mRNA expression indexes in the cancer cells are coded as follows: HB-EGF, filled red circles; AR, filled blue circles; TGFα, filled yellow circles; EGF, open red circles; epiregulin, filled green circles; betacellulin, open blue circles; epigen, open green circles. (B) The amounts of HB-EGF, AR, TGFα and EGF proteins in culture media from breast cancer cell lines cultured for 48 h were measured by ELISA. The cells were counted, and the HB-EGF, AR, TGFα and EGF concentrations were calculated according to the cell numbers. HB-EGF, red bars; AR, blue bars; TGFα, yellow bars; EGF, green bars. The data represent the means ± SD of three independent experiments. *P<0.05, versus the concentrations of the other EGFR ligands.
IJC_25472_sm_suppinfofig4.tif582KSupplementary Figure 4. Expression of HB-EGF mRNA in cancer tissues from patients with TNBC (N=9), overexpression of HER2 (N=15) or breast cancer except for TNBC or overexpression of HER2 (N=21). The expressions of ER, PR and HER2 were scored as described in the Materials and Methods. The HB-EGF mRNA expression indexes were determined by real-time PCR and coded as follows: TNBC, red circles; overexpression of HER2, blue circles; breast cancer except for TNBC or overexpression of HER2, green circles. The horizontal line indicates the mean mRNA expression index for each group. *P<0.05, versus the mRNA expression indexes in cancer tissues from patients with TNBC and breast cancer except for TNBC or overexpression of HER2 using the Bonferroni t-test.
IJC_25472_sm_suppinfofig5.tif1258KSupplementary Figure 5. Weekly alterations in body weight during tumor growth of breast cancer cells in NOD/SCID mice (n=8). (A) MDA-MB-231 cells. (B) BT474 cells. (C) MCF7-HER2 cells. CRM197 (50 mg/kg) was injected intraperitoneally every day for 10 days after the tumor volume exceeded 100 mm3. The data represent the means ± SD of the body weight changes in the 8 mice.
IJC_25472_sm_suppinfofig6.tif851KSupplementary Figure 6. Densitometric analysis of the band intensities for the immunoprecipitation-immunoblotting analyses shown in Figure 3B. (A, B) Heterodimer formation of EGFR with HER2 (A) or HER3 (B) in the presence of recombinant HB-EGF (rHB-EGF). *P<0.05, versus serum-free medium. **P<0.05, versus trastuzumab plus rHB-EGF. ***P<0.05, versus serum-free medium, rHB-EGF or anti-(neuregulin 1) NRG antibody plus rHB-EGF. (C) Heterodimer formation of HER3 with HER2 in the presence of recombinant NRG. (rNRG) *P<0.05, versus rNRG, trastuzumab plus rNRG or CRM197 plus rNRG. (D) Heterodimer formation of HER3 with EGFR in the presence of rNRG. *P<0.05, versus rNRG or trastuzumab plus rNRG. The data represent the means ± SD of three independent experiments.
IJC_25472_sm_suppinfofig7.tif1230KSupplementary Figure 7. Inhibitory effect on the phosphorylation of AKT and ERK in breast cancer cells treated with CRM197 (100 nM) or an anti-HB-EGF neutralizing antibody (1 μg/ml) for 48 h. (A) BT549 cells. (B) MDA-MB-231 cells. (C) MCF7-HER2 cells. The phosphorylation of AKT or ERK and β-actin were evaluated by immunoblotting.
IJC_25472_sm_suppinfofig8.tif1811KSupplementary Figure 8. Inhibitory effect on the phosphorylation of AKT and ERK in breast cancer cells treated with siRNAs for HB-EGF, AR or TGFα for 48 h. (A) BT549 cells. (B) MDA-MB-231 cells. (C) MCF7-HER2 cells. The phosphorylation of AKT or ERK and β-actin were evaluated by immunoblotting.
IJC_25472_sm_suppinfofig9.tif1894KSupplementary Figure 9. Cell characteristics mediated by HB-EGF, EGFR, HER2 or AKT signaling in immortalized MEFs. Wild-type MEFs (Wt), HB-EGF-/- MEFs (HB-/-) or HB-EGF-/- MEFs stably transfected with the human HB-EGF gene (HB-/- + hHB) were infected with retroviruses expressing EGFR, HER2 or AKT, and plated on Matrigel. (A) The protein levels of ErbB3, ErbB4 and β-actin were evaluated by immunoblotting in cells harvested from the respective colonies. (B) The mRNA expressions of EGFR, ErbB2, ErbB3, ErbB4 and GAPDH were analyzed by RT-PCR in cells harvested from the respective colonies.
IJC_25472_sm_suppinfotable1.tif414KSupporting Information Table 1
IJC_25472_sm_suppinfotable2.tif185KSupporting Information Table 2
IJC_25472_sm_suppinfo.doc47KSupporting Information

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