Figure 3: ATM kinase regulates HER2 receptor levels and stability.
From: ATM kinase sustains HER2 tumorigenicity in breast cancer
(a) NeuT levels were revealed by immunohistochemistry on tumours from NeuT mice injected with 10 mg kg−1 KU-55933 in DMSO or with DMSO as in Fig. 2d, scale bars, 30 μm. (b,c) Western blot experiments showing HER2 receptor protein levels in SKBR3 cell line and SKOV3 cell line upon treatment with ATM inhibitor KU-55933 (10 μM for the indicated times). (d) Western blot for detection of HER2 receptor levels in HER2+ breast cancer cell line stably infected with retrovirus expressing control (shR5, labelled −) or ATM RNA interference (shATM#1, labelled +). (e) Representative histograms showing flow cytometry analysis of HER2 expression on cell surface. Incubation with phycoerythrin-conjugated goat anti-mouse IgG alone served as negative controls (isotype, grey solid histograms). MFI, mean fluorescence intensity. (f) Western blot for detection of HER2 and ATM in the indicated cell lines stably infected with retrovirus expressing control (shR5, labelled −) or ATM RNA interference (shATM#1, labelled +). (g) Representative western blot showing HER2 protein degradation upon treatment with KU-55933 (10 μM) for 16 h and subsequently inhibition of protein synthesis with cycloheximide (100 μg ml−1) for indicated times. (h) Representative western blot (upper panel) and quantification (lower panel) of HER2 protein degradation in MCF7-HER2shR5 cell line compared with MCF7-HER2shATM. CHX, cycloheximide (100 μg ml−1).
