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. Author manuscript; available in PMC: 2023 Oct 14.
Published in final edited form as: Oncogene. 2023 Jul 11;42(34):2521–2535. doi: 10.1038/s41388-023-02768-6

Fig. 3. NICD is responsible for the tumorigenic potential of mLPS1 cells.

Fig. 3

A CRISPR targeting strategy to ablate the Rosa-NICDOE cassette in mLPS1 cells. The NICD cDNA containing exon 28 to 34 of Notch1 gene was inserted into the Rosa26 locus. Two guide RNAs (gRNAs) each spanning two exons were designed to target the NICDOE transgene without affecting the endogenous Notch1 gene. The sequencing results validated the correct targeting. B Relative mRNA levels of Notch related genes in mLPS1 and mLPS1 NICD knockout (mLPS1ΔNICD) cells (n = 3). C NICD and GFP protein levels in mLPS1 and mLPS1ΔNICD cells. D Cell proliferation of mLPS1 and mLPS1ΔNICD cells (n = 5). E Colony formation assay of mLPS1 and mLPS1ΔNICD cells (n = 3). Representative pictures of colony size (left) and quantification of colony area (right) were shown. F RT-qPCR analysis of mesenchymal stem cell markers Cd73, Cd90, and Cd105 in mLPS1 and mLPS1ΔNICD cells (n = 3). G Tumorigenicity of mLPS1 and mLPS1ΔNICD cells after subcutaneous transplantation into the left and right flanks of NRG mice, respectively (n = 5). H Images of the grafted tumors after surgical removal from the NRG recipient mice. I The average weights of the tumors (n = 5). Data are presented as mean ± SD, *P < 0.05, **P < 0.001.