Endogenous ferrochelatase during primary in vitro differentiation.

(A) (left) Total RNA was isolated from primary differentiated EBs over a 5-day period. Northern blot analysis was conducted using 15 μg total RNA and probed with a random-primed mouse ferrochelatase cDNA (MFC). The membrane was stripped of the ferrochelatase probe and reprobed with a random-primed human EKLF cDNA (EKLF). Amounts of RNA loaded for each time point were assessed using ethidium bromide staining of 18S and 28S ribosomal RNA (rRNA). (right) Graphical representation of ferrochelatase mRNA levels depicted in Northern blot (left). Values were normalized to rRNA levels using scanning densitometry. (B) Endogenous ferrochelatase protein is demonstrated by immunoblotting. (C) Ferrochelatase promoter transgene expression during primary in vitro differentiation. Transgene expression was determined at each 24-hour time point by disrupting the EBs with brief trypsinization and quantifying EGFP expression in live cells using flow cytometry. Black lines represent transgenes that have −0.150 kb Sp1 elements and the −0.375 kb NF–E2 and GATA sites. Gray lines represent the −0.150 kb transgene that contains only the Sp1 elements, the −0.125 kb transgene that contains no known functional elements, and the transgene-negative cell line BK4.