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. 2024 Dec 11;8(3):e202403122. doi: 10.26508/lsa.202403122

Figure 6. Analysis of NOTCH1 localization and signaling activity upon HCN2 silencing.

Figure 6.

(A) Confocal sections of MCF10A cells treated and immunolabeled as indicated. (A’) HCN2 silencing causes marked NOTCH1 accumulation in the Giantin-positive cis-GA compartment when significantly reducing cell surface NOTCH1 (N1) levels (quantified in (A’)). (B) Confocal sections of MCF10A cells treated and immunolabeled as indicated. (B’) HCN2 silencing markedly depletes the TGN46-positive TGN compartment but not the golgin-97-positive TGN compartment (quantified in (B’)). (C, D, E) The Notch reporter cell line, MCF10A-RbpJk-Luc and RT-qPCR analysis reveal that HCN2 silencing suppresses basal Notch signaling. (F) Western blot analyses indicate that HCN2 depletion reduces both basal and EGTA-stimulated N1ICD levels. (F) Note that levels of N1ICD in unstimulated and EGTA-stimulated cells are not comparable in (F) because of different detection methods (see materials and methods). (G) Western blot analysis indicates that HCN2 silencing results in marked accumulation of N1FL. *, **, ****, and ns indicate P < 0.05, P < 0.01, P < 0.0001, and not significant, respectively.