Figure 6. GATA-3-deficient progenitors express cell cycle inhibitory and pro-apoptotic genes, which block early T cell development.

(A) qPCR for transcripts expressed by DN2 cells sorted after 8 days of co-culture of Gata3^+/− (white bars) and Gata3^−/− (black bars) bone marrow LSKs with OP9-DL1. All samples were normalized to β-actin. Mean and SEM were calculated from 3 independent qPCR analyses. (B) Wild type bone marrow sorted LSKs co-cultured with OP9-DL4 were retrovirally transduced with either MIY or MIY-Cdkn2b vector on day 5. Cells were sorted 24 hours post-transduction as CD45⁺ YFP⁺ CD44⁺CD25⁻ DN1, cultured on OP9-DL4 cells for 3 days. Cultures were analyzed by flow cytometry for expression of CD44 and CD25 as well as (C) Annexin V and PI. (D) Percentage of Annexin V⁺ CD45⁺ cells present in the wells of wild type YFP⁺ DN1 progenitors expressing MIY (white bar) or MIY-Cdkn2b (black bar) co-cultured with OP9-DL4 for 3 days. (E) Wild type bone marrow LSKs were cultured with OP9-DL4 cells for 5 days and DN1 (CD44⁺CD25⁻) or DN2 (CD44⁺CD25⁺) cells purified by flow cytometry. Sorted DN1 or DN2 cells were cultured on OP9-DL4 cells in the presence or absence of PD0332991 (2 μg/ml) and co-cultures analyzed after 3 days for presence of CD44 and CD25 as well as (F) Annexin V+ and PI+ cells. (G) Percentage of Annexin V⁺ CD45⁺ untreated (white bar) or PD0332991-treated (black bar) DN1 and DN2 cells 3 days after the DN1/DN2 sort. All data is represented as mean ± SEM; n = 2, *p < 0.05 (Student t-test).