Figure 1.

Specific inhibition of HER2 signaling in HER2-overexpressing breast cancer cells reduces MIF protein levels. (a) Endogenous HER2 protein levels in human breast cancer cells. Representative immunoblot of cell lysates from the indicated cell lines. mErbB2, murine ErbB2 cell line. Actin, loading control. (b) HER2 inhibition destabilizes endogenous MIF protein. The indicated cells were treated with 2 μM CP724.714 or DMSO for 48 h. Immunoblot. Actin, loading control. (c and d) The specific HER2 inhibitor CP724.714 reduces endogenous MIF levels in a dose- (c) and time- (d) dependent manner. MDA-MB-231 serves as negative control. pAKT and pERK1/2 are functional controls for HER2 inhibition. Immunoblot analyses, Gapdh as loading control. (e) Depletion of HER2 in HER2-overexpressing cells leads to reduced MIF levels. SK-BR-3 cells were transfected with two different siRNA against HER2 (1 and 3) or control siRNA (scr). After 3 days, protein levels were assessed by immunoblots. Gapdh, loading control. (f) In contrast to reduced MIF protein, corresponding MIF mRNA levels remain unchanged after CP724.714 treatment (2 μM). SK-BR-3 cells, qRT-PCR normalized to 36B4. Relative values in (ratio (2^−ddCT)). Error bars indicate S.E.M. of two independent experiments in triplicates each. (g) HER2 inhibition causes growth inhibition in HER2-overexpressing cells. Cells were seeded (day 0) and cultured for 24 h (day 1), then treated with 2 μM of CP724.714 (CP) for 24 h or left untreated, and followed up to day 5. Cell confluence measured daily by CELIGO Cytometer for MDA-MB-453 (***P=0.0006) and SK-BR-3 (**P=0.0037). Error bars indicate the ±S.E.M of two independent experiments in duplicates each. Student's t-test of day 5, one-tailed, P-value: *P<0.05, **P<0.01 and ***P<0.001