Fig. 3

GSK3β and mTORC1 regulate EDI3 expression downstream of PI3K/Akt. A, Overview of key proteins in the HER2 pathway that initiate signalling upon HER2 activation, with the inhibitors used in the present study in red. B, Representative Western blot of pHER2, HER2, EDI3, pAkt, and total Akt protein levels in MCF-7/NeuT cells treated with 5 or 10 µM LY294002 for 7 days. C, mRNA expression of EDI3 (left) and representative Western blot of pHER2, HER2, EDI3, p-mTOR, and mTOR protein levels (right) in MCF-7/NeuT cells treated with 1 or 3 µM everolimus for 3 and 7 days. D-E, mRNA expression of EDI3 (left) and representative Western blot of pHER2, HER2, EDI3, p-mTOR, and mTOR protein levels (right) in D, HCC1954 and E, SKBR3 cells treated with 1 or 3 µM everolimus for 1, 2, and 3 days. F, mRNA expression of EDI3 (left) and representative Western blot of pHER2, HER2, EDI3, and β-catenin protein levels (right) in MCF-7/NeuT cells treated with 1 or 2.5 µM CHIR-99021 for 3 and 7 days. G-H, mRNA expression of EDI3 (left) and representative Western blot of pHER2, HER2, EDI3, and β-catenin protein levels (right) in G, HCC1954 and H, SKBR3 cells treated with 1 or 2.5 µM CHIR-99021 for 1, 2, and 3 days. I-K, EDI3 mRNA expression after inhibiting I, HIF1α with 10 and 20 µM KC7F2, J, CREB with 0.1 and 1 µM compound 3i , or K, STAT3 with 5 and 10 µM C188-9 for 1, 2 and 3 days in SKBR3 cells. Data represent the mean ± SD of at least three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant). * in panels C and F indicate significant difference from untreated control; # represents difference to cells treated with dox alone