Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 in 100 μl RIPA-buffer, quantified by Bradford analysis, mixed with 40 μl loading buffer (4 × SSB, 5% b-mer- captoethanol), denatured, fractionated on a 12.5% SDS- PAGE and electroblotted onto nitrocellulose. The membranes were incubated overnight with rabbit poly- clonal antibodies against PFKFB2 (N-term, AP8146a, Abgent), PFKFB2 phosphorylated Ser466 (University of Dundee, UK) or mouse monoclonal antibodies against a-TUBULIN (DM1A, CalBiochem, Nottingham, UK) as a loading control. Specifically bound antibodies were detected with anti-rabbit, anti-sheep or anti-mouse horseradish-peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Uppsala, Sweden) and visualized by chemiluminescence (ECL, Amersham) and subsequent exposure to AGFA Curix X-ray films for 1 second to 30 minutes. Generation of CCRF-CEM derivatives with doxycycline- induced PFKFB2-15A and -15B expression The lentiviral conditional expression constructs pHR- tetCMV-PFKFB2-15A and -15B (U264 and U265) were generated using the GATEWAY™technology (Invitrogen, Carlsbad, CA). The details of this procedure and the gen- eration of stable clonal cell lines with tetracycline-regulated expression of cDNAs cloned into such constructs has been described previously [30]. In brief, human sequences coding for PFKFB2-15A and -15B mRNA were PCR-amplified using unique SacI-flanked forward primer 5’-GAGCTCT- GTGCTCGACGAGCTCGT-3’ and XbaI-flanked isoform specific reverse primers 5’-TCTAGATGGGTCTTCGGC- TAGT-3’ (for PFKFB2-15A) and 5’-TCTAGAGTA- GATCCCAGTCGT-3’ (for PFKFB2-15B) and cDNA from CEM-C7H2 cells as template. The purified PCR product was cloned into the SacI-XbaI site of pENTR-MCS-deltaN- coI (U243), thereby generating pENTR207-PFKFB2-15A (U260) and pENTR207-PFKFB2-15B (U263). Constructs were sequence-verified and subsequently recombined into the “destination vector” pHR-tetCMV-Dest-IRES-GFP (U192) to generate pHR-tetCMV-PFKFB2-15A-ires-GFP (U264) and pHR-tetCMV-PFKFB2-15B-ires-GFP (U265), as previously described [30]. The lentiviral plasmids were transfected into 293T packaging cells together with pVSV- G and pSPAX (kindly provided by Didier Trono), and the lentivirus-containing supernatants were used to transduce CEM-C7H2-2C8, which constitutively express rtTA [20]. After limiting dilution cloning, three clonal cell lines expressing the PFKFB2-15A isoform, termed CEM- PFKFB2-15A#C3, #D6 and #E8, and three expressing PFKFB2-15B named CEM-PFKFB2-15B#65, #66 and #95, were selected for further experiments. Real time RT-PCR For high through-put real time RT-PCR, 50 μl of diluted cDNA (2 ng/μl) were added to 50 μl of TaqMan Page 4 of 11 Universal MasterMix (Applied Biosystems, Foster City, CA) and introduced into microfluidic cards containing real time RT-PCR mixes for human PFKFB2 (Hs01015408_m1, Applied Biosystems) and TATA box- binding protein for mRNA normalization (TBP, Hs00427620_m1) according to the manufacturer’s guide- lines. After equilibration at RT, the channels were filled with 100 μl of reaction mix and centrifuged two times for 1 minute at 1000 rpm. Thereafter, the cards were sealed, loaded into the HT7900 real time machine (Applied Bio- systems), and run with a 2-step PCR thermo-protocol that included an initial 94.5°C step for 10 min followed by 40 cycles of 97°C for 15 sec alternating with 60°C for 1 min. Fluorescence signal intensities were read during the 60°C temperature step. Similarly, but on conventional 96 well plates, mRNA encoding the 2 PFKFB2 splice variants 15A and 15B were quantified (Hs01015410_m1 and Hs01016554_m1, Applied Biosystems, for PFKFB2- 15A and PFKFB2-15B, respectively). Primary real-time PCR data analysis was performed with SDS software version 2.2.1, and further analysis was performed in R (version 2.8). Data from 3 technically replicated mea- surements were averaged and normalized to the internal TBP control. Log2 fold change values (M values) were calculated for 3 biological replicates by comparing nor- malized real-time PCR data from GC-treated samples against data from the corresponding control samples. M values were averaged for the 3 biological replicates and p-values were calculated (Student’s t-test) to test against the hypothesis of no differential expression (mean M = 0). Results Expression and regulation of PFKFB1, 3 and 4 isoenzymes in lymphocytes and malignant lymphoblasts In a previous study, we showed that PFKFB2 is regu- lated by GC in 11/13 children suffering from T-ALL and preB-ALL [5]. To elucidate whether GC exclusively regulated PFKFB2 in lymphoid cells, or whether other PFKFB-isoenzymes are susceptible to GC-treatment as well, we re-analyzed this data using the more robust GCRMA normalization and included 18 additional ALL patients undergoing GC therapy as well as 3 non-leuke- mic children who received GC as part of their epilepsia treatment, and 6 GC-sensitive and 6 GC-resistant deri- vatives of the CEM-C7H2 T-ALL cell line. Additional file 1, Table S3 summarizes mean expression levels (mE-value) and mean GC regulation (mM-value) of the 3 isoforms, i.e. PFKFB1, PFKFB3 and PFKFB4, in these samples, organized in the following groups: T-ALLs, precursor B-ALLs [as one group and as 3 separate groups based on molecular defects: hyperdiploid, ETV6/ RUNX1 (previously TEL/AML1) translocation, and a heterogeneous group termed “others"], non-leukemic