Fig. 1

Engineering of photo-activatable Notch. Scheme of the engineered system and optogenetic model of action used throughout this study. a The LOV2^WT:Zdk1-N1ICD or LOV2^V416L:Zdk1-N1ICD is anchored in the cell membrane until photo-activation (456 nm, blue light bulb) induces dissociation of the complex, where Zdk1-N1ICD translocates to the nucleus to activate Notch-target genes. b The vector is designed to express LOV2^WT or LOV2^V416L mutant and Zdk1-N1ICD linked by a P2A “self-cleaving” sequence; c resulting proteins containing LOV2^WT/^V416L (with membrane targeting sequence, MTS) and Zdk1-N1ICD. P—CMV promoter, MTS—myristoylation-targeting sequence, LOV2—light-oxygen-voltage-sensing domain 2, P2A—porcine teschovirus-1 2A, Zdk1- Zdark 1, N1ICD—human NOTCH1 intracellular domain. d Schematic representation of blue light activation pattern (pulses) used in optoNotch system. e–g The HEK293T cells expressing either LOV2^WT-Zdk1-N1ICD or LOV2^V416L-Zdk1-N1ICD were transfected by the 12xCSL-Luc reporter and subsequently light activated. The relative luminescence units (RLU) level was measured in LOV2^WT (e) or LOV2^V416L-Zdk1-N1ICD (f) and mock-transfected cells (g) 48 h after activation. The duration of photoactivation of optoNotch (0 h, 1 h, 3 h, and 12 h) does not equate to the increased response. h The mRNA expression of human HES1, HEY1, and NOTCH3 genes was determined by qPCR (2^–∆∆Ct) 16, 24, and 48 h after light stimulation of optoNotch (LOV2^V416L)-HEK293T cells. The results are presented as fold change (FC) values (mean ± SD) normalized to the human GAPDH gene expression. All data were analyzed with a one-way ANOVA test and Tukey’s multiple comparisons post-hoc test vs. not light stimulated cells (the time point 0 h and D; dark, respectively). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 were considered statistically significant, ns – non significant