Colony-forming assays and limit dilution LTCs demonstrate an inhibition of differentiation of murine RAG-1^−/− and wild-type Sca1⁺lin⁻ cells by activated Notch1.

(A) Activated Notch1 leads to a significant decrease of total colonies in Notch1 transduced (MSCV-ICN/GFP and MSCV-Δ E/GFP) RAG-1^−/− Sca1⁺lin⁻ bone marrow cells in comparison to control vector (MSCV-GFP) transduced cells in colony-forming assays. Chart shows average of colonies per 500 cells ± SEM of 4 independent experiments. Data were analyzed using the Student t test (n = 4;P = .048 MSCV-ICN/GFP versus control;P = .003 MSCV-ΔE/GFP versus control). (B) Morphology of colonies of transduced RAG-1^−/−Sca1⁺lin⁻ cells in a colony-forming assay. RAG-1^−/−Sca1⁺lin⁻ bone marrow cells expressing activated Notch1 form fewer and bigger colonies (MSCV-ICN/GFP and MSCV-ΔE/GFP) than control vector-transduced cells (MSCV-GFP). (C) RAG-1^−/−Sca1⁺lin⁻ cells transduced with activated Notch1 show a higher LTC-IC frequency in LTCs with limiting dilutions than control vector-transduced cells. Chart shows average of LTC-ICs per 100 000 cells ± SEM generated from 4 to 5 limiting dilutions (n = 3). Data were analyzed using the Student t test (P = .025 MSCV-ICN/GFP versus control;P = .05 MSCV-ΔE/GFP versus control). (D) LTC-IC frequency per 100 000 cells in wild-type Sca1⁺lin⁻ bone marrow cells expressing activated, Notch1 (MSCV-ICN/GFP) in comparison to control vector-transduced cells (MSCV-GFP; 4 independent experiments, paired t test, P = .03). (E) Single cells picked from LTC-IC colonies were transferred into fresh semisolid medium. In this secondary colony-forming assay, Notch1-transduced cells (MSCV-ICN/GFP) form bigger colonies than control vector-transduced cells (MSCV-GFP). Photos show 2 representative colonies and their fluorescence under UV light.