Confirmation of microarray results by real time RT-PCR analysis
| Gene . | Microarray . | Real time RT-PCR . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 32D . | Ba/F3 . | 32D . | |||||||
| ITD/WT . | IL-3/Med . | FL/Med . | ITD/WT . | P . | IL-3/Med . | FL/Med . | ITD/WT . | D-65476/Med . | |
| Up-regulated | |||||||||
| PIM2 | 998.30 | 352.80 | 1.00 | 42.9 | .001 | 7.7 | 0.51 | 9.73 | 0.052 |
| GP49B | 206.50 | 43.60 | 1.54 | 226.4 | .006 | 61.5 | 12-0.2 | 0.28 | 0.047 |
| SOCS-2 | 3.10 | 3.30 | 0.70 | 8.4 | .009 | 5.4 | 2.1 | 2.32 | 0.72-1.35 |
| SOCS-3 | 18.23 | 31.05 | 4.92 | 6.19 | .009 | 3.3 | 1.7 | 5.83 | 0.042 |
| PGE-R | 3.10 | 2.70 | 0.60 | 1.68 | .019 | 2.47 | 0.71 | 0.4-27.55 | 0.4-1.72 |
| Down-regulated | |||||||||
| maf-B | 0.10 | 0.50 | 1.60 | 0.6 | .004 | 1.8 | 1.4 | 2.07 | 0.7-1.74 |
| PU.1 | 0.60 | 1.10 | 1.30 | 0.31 | .006 | 2.05 | 3.56 | 0.33 | 2.00 |
| RARγ | 0.60 | 1.20 | 1.10 | 0.378 | .011 | 0.71 | 3.46 | 0.04 | 6.3 |
| MAID | 0.66 | 0.53 | .014 | 0.61 | 1.44 | 0.5 | 1.84 | ||
| GADD45 | 0.22 | 0.16 | 0.69 | 0.35 | .028 | 0.18 | 1.61 | 0.12 | 4.2 |
| RB2/p130 | 0.49 | 0.15 | 0.58 | 0.55 | .002 | 0.52 | 1.98 | 0.61 | 3.9 |
| p16 ink 4A | 0.75 | 0.90 | 1.00 | 0.19 | .011 | 6.47 | 5.35 | 77.17 | 2.00 |
| Frat1 | 0.30 | 0.02 | 0.68 | 0.143 | .028 | 0.25 | 1 | 0.34 | 4.7 |
| LSC | 0.40 | 0.90 | 1.00 | 0.37 | .027 | 3.16 | 4.31 | 0.4 | 1.85 |
| RGS2 | 0.30 | 0.30 | 1.00 | 0.33 | .002 | 0.26 | 1.8 | 0.01 | 1.92 |
| Gα15 | 0.60 | 1.10 | 1.20 | 0.265 | .022 | 2.27 | 3.04 | 0.79-1.28 | 1.22 |
| TISS11 | 0.57 | 0.50 | 0.90 | 0.28 | .016 | 1.1 | 3.25 | 0.35 | 1.78 |
| Evi-2 | 0.47 | 1.01 | 1.37 | 0.37 | .005 | 0.47 | 1.31 | 0.66 | 2.54 |
| Gene . | Microarray . | Real time RT-PCR . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 32D . | Ba/F3 . | 32D . | |||||||
| ITD/WT . | IL-3/Med . | FL/Med . | ITD/WT . | P . | IL-3/Med . | FL/Med . | ITD/WT . | D-65476/Med . | |
| Up-regulated | |||||||||
| PIM2 | 998.30 | 352.80 | 1.00 | 42.9 | .001 | 7.7 | 0.51 | 9.73 | 0.052 |
| GP49B | 206.50 | 43.60 | 1.54 | 226.4 | .006 | 61.5 | 12-0.2 | 0.28 | 0.047 |
| SOCS-2 | 3.10 | 3.30 | 0.70 | 8.4 | .009 | 5.4 | 2.1 | 2.32 | 0.72-1.35 |
| SOCS-3 | 18.23 | 31.05 | 4.92 | 6.19 | .009 | 3.3 | 1.7 | 5.83 | 0.042 |
| PGE-R | 3.10 | 2.70 | 0.60 | 1.68 | .019 | 2.47 | 0.71 | 0.4-27.55 | 0.4-1.72 |
| Down-regulated | |||||||||
| maf-B | 0.10 | 0.50 | 1.60 | 0.6 | .004 | 1.8 | 1.4 | 2.07 | 0.7-1.74 |
| PU.1 | 0.60 | 1.10 | 1.30 | 0.31 | .006 | 2.05 | 3.56 | 0.33 | 2.00 |
| RARγ | 0.60 | 1.20 | 1.10 | 0.378 | .011 | 0.71 | 3.46 | 0.04 | 6.3 |
| MAID | 0.66 | 0.53 | .014 | 0.61 | 1.44 | 0.5 | 1.84 | ||
| GADD45 | 0.22 | 0.16 | 0.69 | 0.35 | .028 | 0.18 | 1.61 | 0.12 | 4.2 |
| RB2/p130 | 0.49 | 0.15 | 0.58 | 0.55 | .002 | 0.52 | 1.98 | 0.61 | 3.9 |
| p16 ink 4A | 0.75 | 0.90 | 1.00 | 0.19 | .011 | 6.47 | 5.35 | 77.17 | 2.00 |
| Frat1 | 0.30 | 0.02 | 0.68 | 0.143 | .028 | 0.25 | 1 | 0.34 | 4.7 |
| LSC | 0.40 | 0.90 | 1.00 | 0.37 | .027 | 3.16 | 4.31 | 0.4 | 1.85 |
| RGS2 | 0.30 | 0.30 | 1.00 | 0.33 | .002 | 0.26 | 1.8 | 0.01 | 1.92 |
| Gα15 | 0.60 | 1.10 | 1.20 | 0.265 | .022 | 2.27 | 3.04 | 0.79-1.28 | 1.22 |
| TISS11 | 0.57 | 0.50 | 0.90 | 0.28 | .016 | 1.1 | 3.25 | 0.35 | 1.78 |
| Evi-2 | 0.47 | 1.01 | 1.37 | 0.37 | .005 | 0.47 | 1.31 | 0.66 | 2.54 |
The cell lines 32D and Ba/F3 stably expressing Flt3-WT or Flt3-ITD were cultured under the same conditions as in the microarray experiments depicted in Figure 1. Subsequently the expression of 23 genes showing significant regulation in the microarray experiments was analyzed by real time RT-PCR analyses. The numbers given represent the ratios of glyceraldehyde phosphate dehydrogenase (GAPDH)–normalized expression values (PCR) or of “average difference” values (microarrays). For significance analyses, a Student t test for paired samples on multiple experiments at different time points of FL stimulation was performed. The last column shows the ratio of expression in 32D/Flt3-ITD cells under the influence of 0.5 μM D-65476 compared with controls treated with solvent (0.01% dimethyl sulfoxide [DMSO]). Each number is representative of at least 3 independent experiments.