Figure 4: NICD1 and RBPJ regulate ERBB3 promoter.
From: A genetic cell context-dependent role for ZEB1 in lung cancer
(a) Schematic for the ERBB3 promoter containing a putative RBPJ-binding site. (b) Immunoprecipitation result showing NICD1 bound to RBPJ in H1299 lung cancer cells. H1299 cells were transiently transfected with FLAG-tagged NICD1, which were immunoprecipitated by anti-FLAG. Both IgG immunoprecipitates and 10% non-immunoprecipitated cell lysate were included in the RBPJ western blotting as controls. ChIP assays result showing that both NOTCH1 (c) and RBPJ (d) directly bound to the RBPJ site on ERBB3 promoter in H1299 cells (triplicates; mean plus s.d.). * indicates t-test P<0.05. Mutation of the RBPJ binding site (e) or knockdown of RBPJ (f) suppressed the inhibitory effect of NICD1 on the ERBB3 promoter activity (triplicates; mean plus s.d.). * indicates t-test P<0.05. qPCR of RBPJ (g) and ERBB3 (h) for H322 lung adenocarcinoma cells transfected with control NT non-targeting siRNA or RBPJ siRNAs (triplicates; mean plus s.d.). * indicates t-test P<0.05. (i) Western blotting of NICD1 and Tubulin for lung cancer cells transiently transfected with miR-200c or a control non-targeting microRNA (NT). (j) Western blotting of ZEB1, NICD1, pERBB3, ERBB3 and Tubulin for lung cancer cells transiently transfected with NOTCH1 siRNA or a control non-targeting siRNA (NT).
