STAT1 and IRF-1 activity and B7-H1 expression. (A) TransAM assay of MAPK-regulated transcription factors. (B) TransAM assay of STAT1α transcription factor in nuclear extracts from RPMI 8226 or RPMI 8226/DN-hMyD88 cells incubated for 30 minutes with 500 IU/mL IFN-γ and with or without 1-hour preincubation with 20 μM UO126. (C) JAK2 and STAT1 phosphorylation in RPMI 8226 cells exposed for 30 minutes to 500 IU/mL IFN-γ with or without 1-hour preincubation with 25 μM AG490 or 20 μM UO126. (D) Same as (C) but from a representative CD-138-selected MM sample. (E) Flow cytometric analysis of B7-H1 expression in RPMI 8226 cells transfected with control NS or STAT1 siRNAs. (F) Same as (E) but with 4 CD138-selected MM samples. (G) Western blot analysis of STAT1 expression in a representative CD138-selected MM sample after transfection with control NS or STAT1 siRNAs (H) Western blot analysis of IRF-1 expression after incubation for 24 hours with 500 IU/mL IFN-γ and pre-treated for 1 hour with 20 μM UO126 or 25 μM PD98059. (I) Flow cytometric analysis of B7-H1 expression by RPMI 8226 cells or 3 CD138-selected MM samples incubated for 24 hours with IFN-γ with control NS or IRF-1 siRNA.