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. 2020 Apr 20;1(1):32–47. doi: 10.1158/2643-3230.BCD-19-0028

Figure 3.

Figure 3. ENU induces senescence of mesenchymal stromal cells. A and B, MSCs were treated with varying doses of ENU for 24 hours in vitro. Seven days later, cells were either stained for SA-β-gal or incubated 24 hours with EdU, then fixed and stained. Five and three independent experiments were done for SA-β-gal and EdU, respectively. A senescence-inducing dose of 10 Gy was used as a positive control. C, Quantitative PCR analysis of RNA isolated from control (10% ethanol) and ENU-treated (0.2 mg/mL) MSCs at day 7. Values are presented as a ratio of target mRNA to 18S rRNA. N = 3 independent experiments, each done in triplicate. D–H, Mice were mock-treated (10% ethanol) or ENU-treated (100 mg/kg). One month later, mice were sacrificed and MSCs were isolated. D and E, The CFU-F assay was used to estimate the proliferative and clonogenic potential of MSCs. MSCs were isolated from collagenase-treated compact bones and either directly plated (D) or passaged once (E) before plating. N = 5 and 9 independent experiments for D and E, respectively. F–H, Early passage MSCs (P1-2) were stained for SA-β-gal or harvested for RNA. N = 3 independent experiments. Significant differences between control (no ENU/mock) and experimental were calculated by two-tailed Student t test for A–F. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.G and H, GSEA of MSCs from ENU-treated versus mock-treated mice shows an enrichment of the p53 pathway and specific SASP factors, shown below enrichment plot. I, Equal numbers of Egr1+/+(WT) or Egr1± (CD45.2) and WT B6.SJL competitor cells (CD45.1) were transplanted into lethally irradiated recipients that were mock- or ENU-treated, 3 weeks before transplantation. Ratios of CD45.2 (Egr1+/+ or Egr1+/−) versus competitor (CD45.1) for short-term hematopoietic stem cells (ST-HSC; Lin− Sca1+ Kit+ CD48− CD150−) were calculated 4 months posttransplant. A repopulation ratio of CD45.2 (WT-Egr1+/+) to CD45.1 in mock-treated conditions was set to 1 (expressed as 100%). Without ENU, Egr1+/− ST-HSC showed an average repopulation frequency of 31%; with ENU treatment, repopulation increased to 95% (P = 0.05 by two-tailed Student t test) suggesting that an ENU-treated microenvironment impacts stem cell fitness.

ENU induces senescence of mesenchymal stromal cells. A and B, MSCs were treated with varying doses of ENU for 24 hours in vitro. Seven days later, cells were either stained for SA-β-gal or incubated 24 hours with EdU, then fixed and stained. Five and three independent experiments were done for SA-β-gal and EdU, respectively. A senescence-inducing dose of 10 Gy was used as a positive control. C, Quantitative PCR analysis of RNA isolated from control (10% ethanol) and ENU-treated (0.2 mg/mL) MSCs at day 7. Values are presented as a ratio of target mRNA to 18S rRNA. N = 3 independent experiments, each done in triplicate. D–H, Mice were mock-treated (10% ethanol) or ENU-treated (100 mg/kg). One month later, mice were sacrificed and MSCs were isolated. D and E, The CFU-F assay was used to estimate the proliferative and clonogenic potential of MSCs. MSCs were isolated from collagenase-treated compact bones and either directly plated (D) or passaged once (E) before plating. N = 5 and 9 independent experiments for D and E, respectively. F–H, Early passage MSCs (P1-2) were stained for SA-β-gal or harvested for RNA. N = 3 independent experiments. Significant differences between control (no ENU/mock) and experimental were calculated by two-tailed Student t test for A–F. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.G and H, GSEA of MSCs from ENU-treated versus mock-treated mice shows an enrichment of the p53 pathway and specific SASP factors, shown below enrichment plot. I, Equal numbers of Egr1+/+(WT) or Egr1± (CD45.2) and WT B6.SJL competitor cells (CD45.1) were transplanted into lethally irradiated recipients that were mock- or ENU-treated, 3 weeks before transplantation. Ratios of CD45.2 (Egr1+/+ or Egr1+/−) versus competitor (CD45.1) for short-term hematopoietic stem cells (ST-HSC; Lin− Sca1+ Kit+ CD48− CD150−) were calculated 4 months posttransplant. A repopulation ratio of CD45.2 (WT-Egr1+/+) to CD45.1 in mock-treated conditions was set to 1 (expressed as 100%). Without ENU, Egr1+/− ST-HSC showed an average repopulation frequency of 31%; with ENU treatment, repopulation increased to 95% (P = 0.05 by two-tailed Student t test) suggesting that an ENU-treated microenvironment impacts stem cell fitness.