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. 2018 Jan 5;25(3):600–615. doi: 10.1038/s41418-017-0002-6

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© ADMC Associazione Differenziamento e Morte Cellulare 2018

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Fig. 5 — Sumoylation decreases Notch1 activity and represses the expression of Notch1 target genes. a K1774/1780/1781/1782R GFP-Flag-NICD1 (TSM) cannot be sumoylated as efficiently as wild-type GFP-Flag-NICD1 (WT). The sumoylated form of Notch1 is indicated with an arrow and the unmodified form with an asterisk. n = 3. b Wild-type NICD1 localizes more potently in the nucleus than TSM. COS7 cells were transfected with GFP-Flag-NICD1 (WT) or TSM, and some samples with His-SUMO1 (S1) as indicated. Images were taken with a Zeiss LSM510 META confocal microscope. c TSM increases the activity of the Notch1 signaling pathway. Notch1 activity was measured from HeLa cells by the 12×CSL luciferase reporter reflecting the activity of the Notch1 signaling pathway. The values shown are normalized luciferase units. n = 3. d–f Sumoylation (S1) decreases the expression of Notch (N1) target genes Hes1, Hey1, and Hey2 during heat shock. The relative expression of mRNA was analyzed by quantitative RT-PCR. n = 3. g–i Sumoylation (S1) decreases expression of Notch1 (WT) target genes Hes1, Hey1, and Hey1, whereas TSM increases the expression of Hes1, Hey1, and Hey2. The relative expression of mRNA was analyzed by quantitative RT-PCR. n = 3. j, k SUMO (S1) represses the expression of the indicated Notch target genes during proteotoxic stress in the form of Bortezomib (Brz). The relative expression of mRNA was analyzed by quantitative RT-PCR n = 3