Expression/regulation of PFKFB2 and its splice variants in leukemic cell lines. (A) The cell lines were cultured as indicated in the presence of 10-7M dexamethasone or 0.1% ethanol as vehicle control, and analyzed for mRNA expression of PFKFB2 (using a primer pair recognizing both splice variants) and TBP as control by quantitative RT-PCR on microfluidic cards. TBP-normalized PFKFB2 expression levels (ΔCT) were used to determine GC regulation (ΔΔCT) expressed as mean M-values ± standard deviation. Asterisks indicate p-values of <0.05. PFKFB2 was undetectable in NALM6 (at 2, 6 and 12 hours) and AT-1 cells prior to, but clearly detectable, after GC treatment. Thus, the extent of regulation (ΔΔCT) could not be quantified. Asterisks indicate statistically significant changes of expression (p < 0.05) as calculated by a paired t-test from 3 replicated values. Regulations were considered biologically significant if the extent of differential expression was larger than 2-fold (indicated by the dashed horizontal line M = 1). (B) Regulation of PFKFB2 variants (open circles for PFKFB2-15A, black circles for PFKFB2-15B) after 24 hour exposure to 10-7M dexamethasone is expressed as mean M-value (ΔΔCT) ±SD. Asterisks indicate p < 0.05. In NALM6 cells, PFKFB2-15B was induced by GC as well. However, since the expression level in the ethanol-treated control sample was below the limit of detection, it was not possible to derive an M-value. In the 697/EU-3 cells, PFKFB-15B was undetectable after 24 hour GC exposure, which might be explained by the fact that PFKFB2 levels in 697/EU-3 were at the limit of detection of this assay or suggest a weak down-regulation. (C) Basal expression of the PFKFB2-15A (white bars) and -15B (grey bars) splice variants was detected in the same cell lines using specific Taqman quantitative RT-PCR and normalized to TBP. Expression levels are reported as minus mean ΔCT (ΔCT, CT values obtained with TBP primers minus CT values obtained with PFKFB2-15A or -15B primers, respectively). In AT-1, both splice variants, and in NALM6 the PFKFB2-15A variant, could be detected in the absence of dexamethasone, although at very low levels (presumably due to slightly higher efficiency of these RT-PCRs compared to the RT-PCR for both variants).