Fig. 4.

Senescence-associated decreased migration and scratch closure. a Senescent cells showed a significant reduction in migration using IncuCyte migration (left) as well as modified Boyden chamber assay (right). Left: Different cells in FBS-reduced medium in inserts migrated towards the reservoir plate filled with standard medium. For normalization, the confluence on the bottom was divided by the confluence on the top for each time point and results are shown after 160 h (n = up to 8 biological replicates per group from three independent experiments). Right: Cells were added to the Boyden chamber and incubated for 8 h, fixed, stained with DAPI, and migrated cells were counted. Migration was normalized to the mean of controls (n = 3 biological replicates per group from three independent experiments). b, c Increasing amounts of senescent cells were added to non-senescent controls to observe the influence on scratch closure (0% = only non-senescent solvent controls, 10% = 10% senescent cells + 90% controls, and so on). b Scratch assay was performed using the wound maker and IncuCyte microscope, and the minimal time of 90% scratch closure was determined (n = up to 8 biological replicates per group from four independent experiments). c Representative images at t = 24 h (top right 50% and top bottom 10% 40 µM SM, blue initial scratch and yellow cell-free area). d Genes associated with wound healing and cell motility were tested in 40 µM SM exposed senescent and non-senescent cells. After RNA extraction from MSCs, a RT-qPCR assay was performed and fold regulation of up- or downregulated genes (≥ 2.0 or ≤ -2.0 in combination with p < 0.05, outside grey box) are shown as means (n = three independent experiments). e The migration of healthy MSCs was increased towards conditioned medium from senescent in comparison to that from non-senescent (control) cells. Conditioned medium was added into the reservoir plate (left) or diluted half and half with culture medium (right). For normalization, the confluence on the bottom was divided by the confluence on the top for each time point and results are shown after 188 h (n = up to 8 biological replicates per group from three independent experiments). Data are represented as Tukey boxplots; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Fig. 6