Fig. 3.
Fig. 3. Notch inhibits spontaneous differentiation under normoxic conditions but has no influence on the cell cycle. (A) Relative mRNA levels of Notch target genes Hes1, 3, 5, Hey1 and Hif-1α target gene Vegf in respect to Hmbs , normalised to the respective control condition of mNSCs treated with DAPT or Dll4 and grown in 3% or 21% O2. Red dotted line indicates threshold for relevant changed genes. (B) Representative bright-field images of mNSCs cultured with (+) or without (−) DAPT. mNSCs were cultured 13 days in 3% or 21% of oxygen and treated with DAPT and their respective solvent as a control (DMSO). Scale bar is 50 μm. (C) Cell counts of mNSCs grown in 3% (blue) or 21% O2 (red). Cells were cultured for 13 days with (+) or without (−) DAPT or their respective solvent (DMSO) as control. Cells were counted as Hoechst+ cells per mm2. (D) Survival of mNSCs under physioxia (blue) and normoxia (red) cultured for 13 days. The number of PI+ cells normalised to the total mNSC number is displayed. (E) Quantitative analysis displaying the cellular distribution in the different cell cycle phases. The plot shows the relative distribution of mNSCs grown under physioxia or normoxia and treated with DAPT, Dll4 and their respective controls across the different cell cycle phases G0-G1, S or G2-M in percent. (F) Representative images of mNSCs stained for Tuj1, GFAP and Hoechst. A total of 105 cells were seeded on a coverslip and grown for three days under hypoxic or normoxic conditions in absence or presence of DAPT, soluble or membrane bound Dll4. Scale bar is 50 μm. (G) Quantitative analysis of the relative amount of Tuj1+ or GFAP+ cells normalised to the total number of cells (Hoechst+) and compared to the control condition. *p<0.05, **p<0.01, ***p<0.001 in respect to control condition using unpaired two-sided t-test (A, G); Two-way ANOVA with Bonferroni-adjusted post-hoc t-test **p<0.01, ***p<0.001 in respect to cells grown in 21% O2 (C∼E); ++p<0.01 +++p<0.001 in respect to cells grown without DAPT (C∼E).
International Journal of Stem Cells 2023;16:293-303 https://doi.org/10.15283/ijsc22168
© 2023 International Journal of Stem Cells
