Figure 1. Establishment of R26^CreER/+;Alk1 ^2f/1f pulmonary endothelial cell line, and effective deletion of the Alk1 gene by 4TM treatment.

A. Schematic diagram of the Alk1 wild-type, Alk1^2loxP (2f), and Alk1^1loxP (1f) alleles. Exons and loxP sequences are denoted by boxes and arrowheads, respectively. Locations of primer pairs detecting specific regions containing loxP sequences for the genomic PCR analysis and the probe recognizing the 5′ region of the Alk1 gene which was used for the genomic Southern blot analysis are indicated. Note that exon 5 encodes the transmembrane domain (indicated by asterisk) of the Alk1 gene. B. After two rounds of FACS with DioAcLDL, about 98% of the sorted immortalized cells were positive for DioAcLDL. C and D. The sorted cells exhibited EC-characteristic polygonal shapes and cell-cell contact inhibition at confluency, and they expressed VE-Cadherin at their junctions. E. Expression of EC-specific marker genes including Flk1, Tie2, Eng, and Edn – along with Alk1 and Smad1– was examined by RT-PCR analysis on clonally expanded 2f/1f-pECs (from 5 cells per well). Amongst several isolated clones, clone #28 showed the EC morphology and the high Alk1 and Edn expression levels and was therefore chosen for further study. β-actin was used for the loading control. E. F. After 2 days of 4TM (1µM) treatment, cells were cultured on normal ECM devoid of 4TM. Primers that can detect both 2f and 1f alleles (3F, 3R, and 6R) amplified both 2f and 1f bands from the parental cells but only the 1f band from 4TM-treated cells. The 1f/1f cell population was maintained for 7 days without additional 4TM treatment. G. Genomic Southern analysis shows efficient genotype conversion from 2f/1f to 1f/1f by 2 days of 4TM treatment. H, I. semi quantitative (H) and quantitative RT-PCR (I) analyses reveal an undetectable level of Alk1 mRNA level in the TM-treated Alk1 ^2f/1f ECs. Negative control indicates no reverse transcription reaction. Error bars are SDs (**p<0.01).