Fig. 2

Notch1 enhances NF-κB signaling by sustaining p65 nuclear localization. A Stills from movies at 0, 17, and 240 min of MDA-MB-231/GFP-p65 cells treatment with vehicle, or 10 ng/ml human TNFα, or 80 µm Jagged1, or 10 ng/ml TNFα and 80 µm Jagged1. In all treatment groups with TNFα, the cells were treated for an initial 10 min, and then TNFα was washed out and replaced with minimal media, or with Jagged1 supplemented media. Cells were imaged live for 240 min using an EPI fluorescence microscope for the duration of the treatment, with one image captured every 2.5 min. Scale bar = 10 μm. B Quantification of normalized GFP-p65 nuclear localization over time from experiment in A. Each timepoint shows an average from 45 cells per treatment, from three independent experiments. C Western blot showing the amount of p65 in the cytoplasmic and nuclear fractions of wild type MDA-MB-231 cells treated for 30 min (upper blots) or 4 h (lower blots) with vehicle, or 10 ng/ml TNFα, or 80 μm Jagged1, or 10 ng/ml TNFα and 80 μm Jagged1 (TNFα + Jagged1). In all treatment groups with TNFα, the cells were treated for an initial 10 min, and then TNFα was washed out and replaced with minimal media, or with Jagged1 supplemented media. The experiment was repeated three times and representative western blots are shown. D Quantification of western blots in C where the nuclear p65 signal was normalized to the lamin A/C signal. The graph shows the fold change in nuclear p65 signal for each treatment relative to the control treatment at both time points. Fold changes were averaged from three independent experiments. E MenaINV mRNA expression in wild type MDA-MB-231 (231) cells treated as in C for 1 or 4 h. The experiments were repeated three times. Bars in E show average fold change MenaINV mRNA expression compared to Control at 1 or 4 h. Data in D and E were analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, ****p < 0.0001, n.s. = not significant