Figure 1.
Stage-Specific Effect of Nkx2-1 Overexpression on Derivation of NKX2-1+ Progenitors
(A) Directed differentiation protocol for NKX2-1+ TPs.
(B) Integration schematic of the Nkx2-1 transgene into the HPRT locus.
(C) Schematic of the knockin reporters (BrachyuryGFP and Foxa2hCD4) and rtTA engineered into the iNkx2-1 line.
(D) Intracellular flow cytometry for NKX2-1 in undifferentiated cells with and without 24 hr of Dox treatment.
(E) Immunostaining of undifferentiated iNkx2-1 cells post-24-hr Dox; nuclear counterstain with propidium iodide (PI). Scale bars represent 100 μm.
(F) Intracellular NKX2-1 flow cytometry plots from D14 following 24-hr pulses of Dox added at indicated intermediate stages. Representative of three differentiations.
(G) Kinetics of endogenous Nkx2-1 expression by RT-qPCR following 24-hr staged pulses of Dox. Fold changes relative to undifferentiated cells, error bars represent SD (n = 3 wells from same differentiation). Representative of three independent experiments.
(H) Representative flow cytometry plot of NKX2-1 expression directly post-24-hr Dox.
(I) Averaged flow cytometry data (n = 12 independent experiments) comparing untreated (no Dox) and treated (Dox D6–D7) samples. Error bars represent SEM. ∗∗∗∗p < 0.0001.
(J) Representative immunostaining of Dox-induced and uninduced cultures at D14. Scale bars represent 100 μm.
See also Figure S1.