Fig. 4

Dlk1⁻ cells and Dlk1⁺ cells had similar proliferative capacity. Dlk1⁺ cells and Dlk1⁻ cells derived from Dlk1⁺ cells were sorted using FACS. a Dlk1⁺ or Dlk1⁻ cells were plated in six-well collagen-coated plates at a density of 300 cells per well. After 10–12 days of culture, the colonies were stained with crystal violet, and the number of colonies was determined. b Dlk1⁺ and Dlk1⁻ cells were plated at 1 × 10⁵ cells per well in six-well collagen-coated plates. After 48 h, BrdU was added to cells at a final concentration of 10 μmol/L for 4 h, and then, BrdU labeling was assessed via FACS, and the proportion of BrdU-positive (BrdU⁺) cells was calculated. c Dlk1⁺ and Dlk1⁻ cells were seeded at a density of 1 × 10³ cells per well in 96-well plates. Cell growth was measured daily using CCK8 assays. d The expression levels of cyclin D1, cyclin E1, cyclin A2, and cyclin B1 in Dlk1⁺ cells and Dlk1⁻ cells were measured via quantitative real-time PCR. Representative images from one of three experiments are shown, and the data are shown as the mean ± SEM of three independent experiments (Mann-Whitney test in a and b, one-way ANOVA with post Dunn’s multiple comparisons test in c, two-way ANOVA with post Bonferroni’s multiple comparisons test in d). n.s. not significant