AML-induced senescence in BMSC. (A) BMSC were cultured alone or with primary AML (0.25 × 10⁶; n = 12) or with CD34⁺ HPC (0.25 × 10⁶; n = 7) for 6 days. Nonadherent cells were removed and BMSC were analyzed for SA-βgal. (B) Bar graph representation of SA-βgal⁺ cells from panel A. (C) BMSC were cultured alone or with primary AML (0.25 × 10⁶; n = 10) or with CD34⁺ HPC (0.25 × 10⁶; n = 5) for 6 days. Nonadherent cells were removed and RNA was extracted from the BMSC. RNA was analyzed for IL-6 and IL-8 expression using quantitative reverse transcription PCR. (D) As for panel C, but analyzed for p16 and p21. (E) BMSC were infected with p16 targeted shRNA or control shRNA lentivirus and cultured for 5 days. AML blasts (0.25 × 10⁶; n = 10) or CD34⁺ HPC (0.25 × 10⁶; n = 5) were cocultured with BMSC with control shRNA or on BMSC with p16 shRNA. AML blast number was assessed using a trypan blue exclusion hemocytometer-based count and CD33/CD45⁺ staining using flow cytometry. (F) To confirm the senescent profile of BMSC from (E) nonadherent, cells were removed and BMSC were analyzed for senescence associated SA-βgal (n = 5). The Mann-Whitney U test was used to compare between treatment groups (*P < .05). Each dot on the dot plots represents a different AML or CD34 HPC sample. ns, not significant.