Fig. 2: CD26 expression in MM cell lines.

a, b Flow cytometry with anti-CD26 (rat clone)-FITC (blue histogram) or isotype control IgG₁ (red histogram) was performed in 14 MM cell lines; U266, KMM1, Nakadai, KMS11, KMS12, KMS18, RPMI8226, KMS20, KMS21, KMS26, KMS27, KMS28, KMS34, and IM9. Open histograms to the left of each panel represent iso IgG₁. CD26 expression on a MM cell lines cultured alone (mono-culture) and b MM cell lines co-cultured with human OCs for 72 h is shown. c MM cell lines (U266, KMS18, KMS26, KMS27, and KMS28) cultured alone or recovered from co-culture with OCs were stained with CD138 and CD26 by immunohistochemistry (Original magnification, ×100). All tested MM cell lines expressed CD138 regardless of mono-culture or co-culture (data not shown) with OCs. In contrast, MM cell lines cultured alone lacked CD26 expression. However, after co-culture with OCs, CD26 expression was upregulated in MM cell lines. Moreover, when subsequently cultured alone for 48 h after removal of co-culture with OCs, CD26 expression in MM cell lines was downregulated again. OCs, both cultured alone and recovered from co-culture, expressed CD26 (TRAP; red-stained, CD26; gray-stained) (original magnification, ×100). d CD26 expression in U266, KMS18, KMS26, KMS27, and KMS28 cultured alone (mono-culture) or co-cultured with OCs was examined by immunoblot analysis. Cell lysates were harvested from each cell lines. β-actin was blotted as a loading control. e The level of CD26/DPPIV derived from supernatants in mono-culture of OCs or MM cell lines and those in the co-culture of OCs with MM cell lines was measured by ELISA. The CD26/DPPIV level in supernatants from the mono-culture of OCs or the co-culture of MM cell lines with OCs was significantly elevated compared with those from the mono-culture of MM cell lines. The data represent the mean ± SE of triplicate wells from the representative of three independent experiments. The error bars represent the range (*p < 0.05. **p < 0.01). f The expression of target genes (CD138, CD26, and TRAP) in MM cell lines (KMS18, KMS26, KMS27, and KMS28) co-cultured with OCs or cultured alone was assayed by real-time quantitative RT-PCR using specific primers. CD26 mRNA expression in MM cells co-cultured with OCs revealed 7- to 19.4-fold increase compared with those co-cultured alone. The results are shown as ratio of indicated gene mRNA/β-actin mRNA. Data represent the the mean ± SE. n = 3. *p < 0.05