Notch-1 regulates IGF-1R expression. (a) Representative Western blot of the indicated proteins in A549 cells trasfected with a control plasmid (cont, left) or with a plasmid expressing Notch-1IC (N1IC), or transfected with a control siRNA (cont, right) or with a siRNA targeting the Notch-1 mRNA (siN1). (b) quantitative RT-PCR of the IGF-1R mRNA in A549 transfected with the nucleic acids described above. Additionally, A549 were exposed to DMSO (C) or with 20 μM MRK-003 (GSI) for 48 hr under hypoxia. mRNA values were normalized for 18S rRNA expression levels. *: P< 0.001; **: P< 0.001; ***: P< 0.01. Columns represent the average of three independent experiments. Error bars represent SD. (c) Western blot analysis of the indicated proteins. H1755 and H1299 cells were either transfected with a control plasmid (N1IC −) or with a plasmid expressing Notch-1IC (N1IC +). The two panels for IGF-1R represent two different exposure times of the same gel. Note increased IGF-1R expression when Notch-1IC was overexpressed. Additional abbreviations: TM, transmembrane, uncleaved Notch-1; IC, Notch-1IC. (d) Western blot analysis of the indicated proteins of different ACL lines (indicated) exposed to 20 μM MRK-003 (GSI+) or to vehicle alone (GSI−) for 48 hr. (e) A549-TR-DEST (lanes 1 and 2) and A549-TR-N1 (lanes 3 and 4) were exposed to either 1 μg/ml doxycycline (DOX), 20 μM MRK-003 (GSI) or both for 48 hr. Note that doxycycline treatment alone reduced the IGF-1R expression levels. A more marked effect was elicited by MRK-003 treatment on the expression of the IGF-1R. However, induction (obtained through doxycycline treatment) of Notch-1IC led to a 4.8-fold increase in the IGF-1R expression levels (compare lanes 3 and 4). Notch-1IC failed to restore the IGF-1R expression levels completely possibly because of the compounding doxycycline effects (compare lanes 1 and 2). (f) Artificial downregulation of HIF-1α causes reduced expression of the IGF-1R in ACL cells under hypoxia. A549 cells were transfected with either a control siRNA or with a siRNA targeting the HIF-1α mRNA. 48 hr after transfection cells were assayed by Western blot analysis for the indicated proteins.