Fig. 4: Irradiation-, replication- and ageing-induced MSC senescence alleviates MSC-induced suppression of MM cell growth in vitro.

A Human MSCs from young non-cancer controls were irradiated at 60 Gy and β-gal activity (blue) was evaluated after 10 days and compared with non-irradiated cells. B β-gal-positive cells were quantitated in irradiated and non-irradiated cultures relative to total cell number (identified by DAPI co-stain). C Expression of CDKN1A was assessed in irradiated and non-irradiated MSCs and normalised to ACTB. D Irradiated MSCs (day 7) from young non-cancer controls and donor-matched non-irradiated controls were cocultured with luciferase expressing KMM1 cells. After 3 days, the relative number of KMM1 cells was enumerated using BLI. E MSCs from eight-week-old C57BL/KaLwRij mice were irradiated at 60 Gy and β-gal activity (blue) was evaluated after 4 days and compared with donor-matched non-irradiated cells. F β-gal-positive cells were quantitated relative to total DAPI-positive cell number. G Expression of Cdkn1a was assessed, normalised to Gapdh. H Irradiated and non-irradiated C57BL/KaLwRij MSCs were seeded and, after adhering overnight, coculture was initiated with luciferase-expressing 5TGM1 cells. After 3 days, the relative number of 5TGM1 cells was enumerated by BLI. I Human MSCs cultures, isolated from young non-cancer controls were passaged twice weekly until they reached replicative senescence (passage 13–15), as demonstrated by assessment of β-gal activity. J Expression of CDKN2A, normalised to ACTB, was analysed at passage 3 and at the passage of senescence (passage 13–15) in MSCs from young non-cancer controls. K MSCs from young non-cancer controls were cocultured with luciferase-expressing KMM1 cells for 3 days and the relative number of KMM1 cells was enumerated using BLI. L β-gal activity was assessed in MSC cultures from eight-week-old C57BL/KaLwRij mice at passage 4 and passage 6. M Expression of Cdkn2a, normalised to Gapdh, was analysed in C57BL/KaLwRij mouse MSCs. N Luciferase-expressing 5TGM1 cells were cocultured with mouse MSCs at passage 4, 5 and 6 and, after 3 days, 5TGM1 cells were enumerated using BLI. O MSCs were isolated by plastic adherence from long bones from young (eight-week-old) and aged (18-month-old) C57BL/KaLwRij mice, cultured for two weeks in vitro and cells were stained for β-gal activity at passage 3. P The percentage of β-gal-positive cells, was calculated relative to DAPI-positive cell number. Q Expression of Cdkn2a, normalised to Gapdh, was analysed in mouse MSCs from young and aged donors (n = 3–4 donors/group). R Mouse MSCs from young and aged donors were cocultured with luciferase-expressing 5TGM1 MM PCs for 3 days and 5TGM1 cells were quantitated by BLI. Representative images of β-gal-stained cells are shown (A, E, I, L, O). Scale bar: 200 µm. Graphs depict mean ± SEM of three (C, D, G, P–R), four (F, H, J, M, N, Q), five (B) or six (K) independent donors. p values are shown for paired t test (B–D, F-H, J, K, M), one-way ANOVA with Holm-Šídák’s post-test (N) or unpaired t test (P–R).