Fig 1: Expression of Notch receptors and DLL4 ligand in BCa cell lines.(A) Relative mRNA expression (normalized to housekeeping gene β-actin) of target genes by qRT-PCR in different BCa cell lines; # significance versus non-invasive, papillary RT-4 cells (G1); * significance between G2-G4 cell lines; p ≤ 0.05, One-way ANOVA, Tukey’s multiple comparisons test, mean + SD, n = 3. (B,C) Protein expression of targets by immunofluorescence in different cell lines. (B) Representative immunofluorescence images of Notch1, Notch2, Notch3, Notch4 and DLL4 staining, exemplarily shown for RT-4 cells; scale bar: 20 µm. (C) Quantification of mean fluorescence intensity in CK AE-positive ROIs; # significance versus non-invasive, papillary RT-4 cells (G1); * significance between G2-G4 cell lines; p ≤ 0.05, One-way ANOVA, Tukey’s multiple comparisons test, mean + SD, n = 3.
Fig 2: Interactions between Notch2 and Notch3 receptors and DLL4 ligand in BCa cell lines.(A) Visualization of co-localization between DLL4 and Notch2/3 receptors by PLA, exemplarily shown for each grade. Prominent fluorescent spots indicate receptor-ligand interactions. Notch2/3-DLL4 complexes were frequently observed in the (peri-) nuclear region, especially in RT-112, T-24 and CAL-29 cells. Notch2/3 complexes (red); plasma membrane (green); nuclei (blue). Scale bar: 10 µm (B,C) Quantification of PLA signal density by particle analyses at single cell level. (B) Sums of particles were normalized to the area of cells (ROIs). The amount of Notch2/3-DLL4 complexes was significantly higher in RT-112 and CAL-29 cells (vs. RT-4, 647-V T-24, KU-19-19). Low grade RT-4 and 647-V (G2) showed moderate Notch2/3-DLL4 levels, while T-24 and KU-19-19 exhibited equally low PLA signal densities. (C) Percentage of interactions complexes with (peri-) nuclear localization. PLA signals visible within DAPI-positive ROIs were normalized to total amount of signals. * Significance between G2-G4 cell lines; p ≤ 0.05, One-way ANOVA, Tukey’s multiple comparisons test; mean + SD, n = 3.
Fig 3: Response to recombinant human DLL4 (rhDLL4) in BCa cells.(A-C) Screening on canonical Notch pathway activation by quantification of HES1 and HEY1 gene expression after 6, 16 h of 250 ng/ml rhDLL4 (A); and quantification of cell viability (B) and proliferation (C) after 72 h of rhDLL4 incubation using different concentrations. RT-4, 647-V and T-24 exhibited increases in HES1/HEY1 gene expression and enhanced cell viability/proliferation after rhDLL4, while RT-112 and CAL-29 showed no DLL4 response in both readouts; * significant differences to vehicle controls, p ≤ 0.05, Tukey’s multiple comparisons test; mean + SD. (C) Notch2/Notch3-DLL4 receptor interactions in rhDLL4-responding cells. Quantification of PLA signal density by particle analyses after 15 min stimulation with 250 ng/ml rhDLL4. Incubation with DLL4 resulted in significantly enhanced interaction complexes in RT-4, T-24 and KU-19-19, especially for Notch3-DLL4; * significant differences to vehicle controls, p ≤ 0.05, Mann-Whitney test; mean + SD.
Supplier Page from Thermo Fisher Scientific for Recombinant Human DLL4 Fc Chimera Protein