Figure 3. Developmental effects of GATA-3 knockdown.

A. Sorted FL precursors were transduced with G3-3W or control (left panels) or nucleofected with Gata-3 targeted or control siRNAs (right panels), and cultured on OP9- DL1 for 3.5 days. Numbers below plots: quadrant percentages. Data shown are representative of two retroviral infection and two nucleofection experiments.

B. Fold change in cell numbers for FL or FLDN subsets over a 3.5 day culture period is shown. (Left panel) FL precursors treated as in Fig. 3A and cocultured with OP9-DL1 or OP9-control as in Fig. 2G. No significant differences in recovery were seen between G3-3W transduced and control cells (two independent experiments). (Right panel) Cell yields from FLDN generated as in Fig. 1E, then infected with control or G3-3W, sorted to pure vector-positive DN1, DN2, or DN3 subsets, then re-plated on OP9-DL1 for 5 to 7 more days. In three experiments, fold differences in yield of G3-3W vs. controls were significant for FLDN1 (p=0.007) and FLDN3 (p=0.007). In three similar experiments with Bcl2-transgenic FLDN, G3-3W transduced samples yielded only 20±3% of the number of cells from control transduced samples of FLDN1, DN2, and DN3 (p=0.004, 0.006, and 0.02 respectively).C. Developmental progression in FLDN subsets. FLDN cells were infected with control or G3-3W vectors, then sorted into purified subsets, returned to OP9-DL1 stroma for 5 to 7 days, then analyzed. Numbers below plots are quadrant percentages. Data shown for DN2 precursors are representative of three experiments in which some “breakthrough” was seen, albeit in DN2 only; in three experiments there was no breakthrough.

D. Bcl-2 transgenic fetal thymic E14-14.5 ETP (“Bcl-2 tg FT”) and nontransgenic ETP (“FT DN1”) were sorted post infection with G3-3W or empty vector and cultured on OP9-DL1 for 3.5 days (representative of two experiments, in which Bcl-2 transgenic and non-transgenic cells were cultured in parallel). Bcl-2 consistently enhanced yields but not progression.

E. A separate experiment from D, but in this case cells were cultured 6.5 days on OP9 DL-1 (representative of two experiments).