CD86 mRNA was up-regulated transcriptionally by SB treatment.

(A) RNA synthesis inhibition assay. The detailed condition of pretreatment with SB and ActD was described in “Materials and methods.” After the addition of ActD for 30 minutes, RNA was extracted from cells after additional incubation in the presence or absence of SB for 1, 3, 6, and 10 hours. The amount of CD86 mRNA was normalized by the amount of GAPDH mRNA; both were obtained from real-time PCR. The relative amounts of normalized CD86 mRNA are presented. (B) Nuclear run-on assay. HL60 cells treated in the presence or absence of 1 mmol/L SB for 3 hours were subjected to the nuclear run-on assay. The membranes, on which full-length cDNAs of CD86 and GAPDH were dot-blotted, were hybridized with in vitro synthesized RNA. The intensity of the CD86 dot-hybridized with SB-treated, radio-labeled de novo RNA was 1.68-fold higher than that of the CD86 dot-hybridized with untreated, radio-labeled de novo RNA. The intensity of GAPDH dots was not altered by SB treatment (1.07-fold).