CD38 expression affected cellular NAD⁺ levels and KCL-22 cell relapse.

<p>(A) A representative flow cytometry profile showed dramatic increase of CD38 expression in KCL-22 cells in response to 1 µM ATRA for 24 h. (B) NAD⁺ levels in KCL-22 cells treated with 1 µM ATRA for 24 h. (C) CD38 levels affected CML cell relapse. Top: KCL-22 cells were treated with 1 µM ATRA for 24 h, and cells were sorted for high vs low/- CD38 expression. Bottom: The sorted cells were used for relapse assay with 2.5 µM IM. For comparison, KCL-22 cells without ATRA treatment (untreated) and ATRA pretreated but not sorted cells (ATRA pretreated) were also analyzed simultaneously. (D) Ectopic CD38 expression in KCL-22 cells. Cells were transduced with a PITA lentiviral vector for CD38 over-expression (CD38 OE) or the empty vector PITA-puro as control (CTL). Transduced cells were enriched by puromycin selection and analyzed by flow cytometry. (E) KCL-22 cells with high ectopic CD38 expression were sorted from the gate R5 of the lower panel in (D), and used for analysis of NAD⁺. The unsorted, empty PITA-puro vector transduced cells were used as control. (F) Apoptosis analysis of high ectopic CD38 expressing cells vs control as described in (E) with and without IM treatment. (G) Relapse assay with 2.5 µM IM for high ectopic CD38 expressing cells (CD38 OE) vs control (puro). For NAD⁺ rescue, 50 µM NAD⁺ was supplied at the beginning with IM. (H) Soft agar colony formation assay. Left: 1 million cells per well were seeded with 2.5 µM IM in the presence or absence of 50 µM NAD⁺. Right: 500 cells per well were seeded without IM in the presence or absence of 50 µM NAD⁺.