DNAM-1 (PVR) and NKG2D (MICA and MICB) ligands are up-regulated on doxorubicin- or melphalan-induced senescent MM cells. (A) SKO-007(J3) cells were incubated with doxorubicin (0.05 μM) or melphalan (22 μM) for 48 hours and left for an additional 24 hours in the absence of the drug. MM cells were then fixed, and incubated overnight at 37°C without CO₂ with SA-β-Gal stain solution (“Senescence-associated β-galactosidase staining”). Senescent cells were identified as blue-stained cells by microscopy. Results are representative of 1 of 3 independent experiments. (B) SKO-007(J3) cells were treated for 24 and 48 hours with doxorubicin or melphalan, then fixed and stained with PI to analyze cell distribution among the different cell-cycle phases. (C) To correlate PVR, MICA, and MICB up-regulation with a specific cell-cycle phase, the untreated and treated SKO-007(J3) cells were incubated with PI and stained for these ligands. The analysis was performed by flow cytometry. The corresponding MFI values of the ligand expression obtained by subtracting the MFI of the isotype control antibody are reported in the table. Results are representative of 1 of 5 independent experiments.