B7-H1 mRNA expression and signal transduction analysis in MM plasma cells. (A) B7-H1 mRNA levels in RPMI 8226 MM cells measured by real-tie polymerase chain reaction after exposure to 500 IU/mL IFN-γ for 6 hours with or without 10 μg/mL cycloheximide or 10 μg/mL actinomycin D (ActD) for 90 minutes. (B) Time-course analysis of B7-H1 mRNA levels after addition of 500 IU/mL IFN-γ to RPMI 8226 MM cells. (C) Flow cytometric analysis of B7-H1 expression in control RPMI 8226 MM cells (set at 100%) and after 24-hour incubation with IFN-γ with or without 1 hour pre-treatment with signal transduction inhibitors, ie, 20 μM UO126 (MEK1/2), 25 μM PD98059 (MEK1), 25 μM SP600125 (JNK), 25 μM AG490 (JAK2), 25 μM LY294002 (PI3K), 3 μM SB203850 (p38MAPK), and 25 μM SN50 (NFκB). (D) Same as (C) but with 5 MM CD138-selected cell samples. (E) Western blot analysis of B7-H1 levels with N20 polyclonal anti-B7-H1 antibody in 2 MM patient CD138-selected plasma cell samples exposed to 500 IU/mL IFN-γ with or without 1-hour preincubation with 20 μM UO126. (F) B7-H1 mRNA levels in 25 MM CD138-selected cell samples exposed for 24 hours to 500 IU IFN-γ with or without 1-hour pre-treatment with 20 μM UO126.