Cytokine profiling shows that IL-6 is the major soluble factor downregulating CD38 expression on MM cells. (A) Four MM cell lines were cultured with control culture medium or culture medium containing the indicated cytokines (IL-6, MIP-1α, SDF-1α, IL-1β, IL-8, IGF-1, TGF-β, OSM, LIF, IL-10, IL-2, IFN-β, IFN-γ) for 72 hours. CD38 expression was measured by flow cytometry after 72-hour incubation, and relative CD38 MFI was calculated in comparison with MFI of control. Heat map was created based on relative CD38 MFI as indicated in supplemental Table 5. (B) Four MM cell lines were treated for 72 hours with IL-6 (0.01-30 ng/mL), and CD38 expression was measured by flow cytometry. (C) RPMI 8226 cells were cultured with control culture media, 2 different BMSC-sup (BMSC-sup #1 or #2), or IL-6 (5 ng/mL) for 12, 24, 48, or 72 hours, and CD38 mRNA level was measured by qRT-PCR. (D) CD138⁺ primary MM cells separated from BMMCs of 5 MM patients using CD138 magnetic-activated cell separation beads were cultured with or without IL-6 (5 ng/mL), and the CD38 mRNA level was measured by qRT-PCR. (E-F) RPMI 8226 (E) and MOLP-8 (F) cells were cultured with control culture medium or IL-6 (5 ng/mL), in the absence or presence of CHX (10 µg/mL) for 6 hours or 12 hours, and the CD38 mRNA level was measured by qRT-PCR. Data are shown as mean plus or minus standard error of the mean. *P < .05; **P < .01; ***P < .001.