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. 2015 Aug 12;89(21):10774–10785. doi: 10.1128/JVI.01463-15

FIG 7.

FIG 7

E4-ORF1 promotes constitutive Myc protein expression in MCF10A cells by a mechanism that requires the activities of EGFR, InsR/IGF1R, and PI3K. (A) E4-ORF1 prevents Myc downregulation in MCF10A cells incubated in serum and growth factor-depleted medium. The indicated MCF10A lines cultured for 24 h in complete medium (CM) were incubated in minimal medium (MM) for the indicated times, when cell extracts were prepared and immunoblotted for the indicated proteins. (B) E4-ORF1 also prevents Myc downregulation in MCF10A cells cultured for 72 h in complete medium. Extracts of the indicated MCF10A lines cultured for 72 h in complete medium were immunoblotted for the indicated proteins. (C) InsR/IGF1R, EGFR, PI3K, and Mek1/2 activities are required for E4-ORF1-induced constitutive Myc expression in MCF10A cells incubated in serum and growth factor-depleted medium. wtORF1 cells cultured in complete medium were incubated in minimal medium containing DMSO, AG1024 (1024) (50 μM), AG1478 (1478) (0.5 μM), LY294002 (LY) (100 μM), or U0126 (10 μM) for 3 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (D) The activities of InsR/IGF1R and PI3K, but not EGFR, are required for Myc expression in MCF10A cells cultured in complete medium. The indicated MCF10A lines cultured in complete medium were treated with DMSO, AG1024 (50 μM), AG1478 (0.5 μM), LY294002 (100 μM), or U0126 (10 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (E) AG1478 or U1026 treatment for 1 h reduces E4-ORF1-induced constitutive Myc expression in wtORF1 cells incubated in serum and growth factor-depleted medium. Extracts of wtORF1 cells cultured in complete medium were incubated for 3 h in minimal medium containing DMSO, AG1478 (0.5 μM), or U0126 (10 μM) during the last 1 h of incubation, at which time the cell extracts were prepared and immunoblotted for the indicated proteins. (F) E4-ORF1-induced constitutive Myc expression depends on Dlg1. At 72 h post-cell plating in complete medium, extracts of wtORF1 cells expressing the Dlg1 shRNA (+) or control scrambled shRNA (−) were prepared and immunoblotted for the indicated proteins.