PRDX2 inhibits proliferation and clonogenic growth of myeloid cells. (A) The myeloid progenitor cell line 32D was transduced with PRDX2-expressing retrovirus. A total of 2 × 104 32D cells overexpressing Prdx2 were deprived of IL-3 and serum (0.5% FCS) for 12 hours in 200 μL of medium in a 96-well plate. Subsequently, cells were placed in medium with 10% FCS or supplemented with IL-3 (0.1 ng/mL). After an 8-hour incubation period at 37°C, 1μCi (0.037 MBq) 3H-thymidine was added to each well, and cells were incubated for an additional 12 hours. Cells were harvested onto glass fiber filters, and emission of bound DNA was analyzed in a scintillation counter. Cells overexpressing PRDX2 proliferated much slower than the vector control cells as measured by 3H-thymidine incorporation assay. The results shown are the mean of triplicates of 3 independent experiments ± SD. The * indicates the significance, calculated by t test; **P < .001 and *P < .05. (B) Colony growth in IL-3–supplemented methylcellulose was reduced in PRDX2-overexpressing 32D cells compared with empty vector control cells. The results represent the mean of 3 independent experiments ± SD (*P < .001, t test). (C) Expression of PRDX2 in 32D cells was confirmed by Western blot analysis. (D) ROSs were measured by the redox-sensitive fluorochrome Dihydrorhodamine 6G and visualized with flow cytometry. Increases in ROS levels after IL-3 stimulation were observed in control vector-transduced cells but not in PRDX2-expressing 32D cells. The result shown here is a representative of 3 independent experiments. (E) Intracellular signal transduction events were analyzed by Western blot analysis with phosphor-specific or total ERK Ab. Phosphorylation of ERK on IL-3 exposure was inhibited by PRDX2 expression. (F) Prdx2 expression was suppressed by shRNA in murine 32D cells. Sufficient inhibition of Prdx2 protein was achieved with 2 shRNA oligonucleotides or a mix of 3 RNAi sequences. Western blot analysis shows reduction in Prdx2 protein level after shRNA transfection (top panel). ROS levels were increased after shRNA-mediated knockdown of Prdx2 in 32D cells as analyzed by flow cytometry (FACS) with the use of the same probe as described above (bottom). Experiment was biologically repeated 5 times. The significance was calculated with the t test (*P < .05). (G) The decrease of Prdx2 expression enhanced proliferation of 32D cells. The results shown are the mean ± SD of 3 independent experiments (*P < .05, Mann-Whitney U test).
