Fig. 1.

Maternal exposure to fine PM_2.5 during pregnancy leads to the progressive senescence of HSCs in offspring of 6 months. a For the colony assay, BM cells (2 × 10⁴ per dish) in PM_2.5-exposed offspring of 6 months were incubated in a methylcellulose-based medium for 12 days and the colonies formed were counted. Representative data are shown for three independent experiments. b For long-term competitive repopulating activity, equal numbers (5 × 10⁵) of BM cells from control or PM_2.5-exposed offspring were co-transplanted with those from competitor mice (CD45.1) into lethally irradiated recipient mice (CD45.1/2, 1000 rads, n = 7) that were also transplanted with BM cells (1 × 10⁶) of CD45.1/2 mice after the first transplantation. PB was collected from the recipient mice at 4 months post-transplantation and the ratio of CD45.1/CD45.2 was assessed by flow cytometry. Donor-derived HSCs engrafted in the BM of the secondary recipient mice were measured by flow cytometry after the procedure of lineage cell depletion (n = 4). c Levels of mitochondrial superoxide anions in the BM HSCs of offspring of 6 months were measured with MitoSOX™ Red reagent using flow cytometry (n = 11). Levels of Nrf2 (d) and p38 phosphorylation (e) were analyzed in the BM HSCs of the offspring after the fixation and permeabilzation procedure (n = 7). f SA-β-gal activity in BM HSCs of the offspring were measured using incubating the cells with C₁₂FDG, a β-galactosidase substrate (n = 11). g Levels of γ-H2AX were analyzed in the BM HSCs of the offspring after the fixation and permeabilization procedure (n = 5). All data are presented as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control, as determined by Student’s t tests