Figure 2. Specificity and efficiency of GATA-3 knockdown.

A. Map of G3-3W cloned in Banshee, showing Gata3-targeting shRNA sequences.

B. Specificity of intracellular flow cytometric staining in Adh.2C2 pro-T cells, in comparison with RAW264.7 myeloid cells (negative control). For additional independent experiments on Adh.2C2 cells see ref. (43).

C. Stable GATA-3 knockdown in Adh.2C2 cells after infection with G3-3W or control vector at 20h and 136h (upper panels). (Lower panels) Correlation of GFP reporter with GATA-3 knockdown: Intracellular GATA-3 staining of Adh.2C2 cells at 65h post infection with G3-3W or control vector (also see ref. (43)).

D. Time courses of shRNA effects on Gata3 RNA levels (left graph, qPCR) and GATA-3 protein levels in Adh.2C2 cells (right graph, intracellular staining). Values from GFP⁺ G3-3W transduced cells, compared to GFP⁺ empty vector controls at the same timepoints, are shown. RNA levels are normalized based on β-actin. For another independent experiment see panel F (representative of at least 3 experiments); similar results for primary cells are shown in Fig. 5C,D.

E. Subset-specific effects of G3-3W shRNA on GATA-3 protein in FLDN cells. FLDN1, DN2 and DN3 subsets from OP9-DL1 culture were infected with G3-3W or control vector, then returned to culture, and intracellular flow cytometric staining was used to measure GATA-3 at 6 days post transduction.

F. Effects of GATA-3 knockdown on Adh.2C2 cells after 6 days. (Top) Gata3 RNA levels in GFP⁺ cells. (Middle) Viabilities, measured by 7AAD exclusion, of GFP⁻ and GFP⁺ fractions of G3-3W infected or control infected cells. (Bottom) Fold changes in cell numbers (same cell populations used in viability analysis; value of 1 = no change). Results of one experiment with Adh.2C2 cells.

G. Comparison of GATA-3 knockdown effects on cell recovery in T-lineage and non-T lineage cells. Maintenance of GFP+ populations (as % of total) after transduction with G3-3W (Gata-3 shRNA) or control constructs is shown for Adh.2C2 cells tracked over 238h culture (top), in primary E13.5 FL precursors (FLP) cultured for up to 112 hr in T-cell conditions on OP9-DL1 (middle), and in FLP cultured in non-T cell conditions on OP9-control stroma (lower). Inhibitory effects shown in 2F and 2G were also representative of results in primary FL-derived precursors growing in T-cell conditions (7 experiments) and FLDNs recultured (7 experiments), although the 2C2 results shown are from a single experiment.