Pecam 1

Liver cells were sorted from mixed, hepatocyte, and non-parenchymal cell
fractions on an Aria Fusion I using a 100 μm nozzle. Cells from the HCC
samples were not fractionated and were sorted directly after tissue digestion.
Zombie Green (Biolegend) was used as a viability dye. Cells were stained with
human specific antibodies against CD45 (Biolegend), PECAM1 (Biolegend), CD34
(Biolegend), CLEC4G (R&D systems), ASGR1 (BD Biosciences), EPCAM
(R&D systems), and TROP2 (Biolegend). Organoids were stained with
antibodies against EPCAM and TROP2. For the humanized mouse samples, cells were
stained either with antibodies against ASGR1 and PECAM1 or human HLA-ABC (BD
Biosciences) and mouse H2-Kb (BD Biosciences). Viable cells were sorted in an
unbiased fashion or from specific populations based on the expression of markers
into the wells of 384 well plates containing lysis buffer.

Tissue and cell permeabilization were performed by incubation with 0.5% TritonX-100 overnight and 0.2% TritonX-100 respectively and blocked with 10% normal goat serum/1% BSA. Inner curvatures of aortic arch were incubated with primary antibodies (CD106 (VCAM), 553330; BD Biosciences and MCP-1, ab7202; Abcam) and descending aorta segments were incubated with primary antibodies (PlxnD1 (PA5-21605; ThermoFisherScientific) and PECAM-1 (553369; BD Biosciences) before incubation with Alexa Fluor 488– and Alexa Fluor 568–conjugated secondary antibodies (1:100; Invitrogen). Cells subjected to flow or tissues were incubated at 4°C overnight in beta catenin (610153; BD Biosciences) followed by 1 hr incubation of Alexa Fluor 488-conjugated phallodin (Invitrogen) at RT and DAPI (Invitrogen). Tissues were mounted en face with Prolong Gold Antifade mountant (Invitrogen) for confocal imaging using Olympus FluoView3000.

Tissue collection, 4% paraformaldehyde fixation, processing, paraffin embedding, whole mount staining for β-galactosidase, and H&E staining were performed using established protocols (Lindsley et al., 2007 (link); Snider et al., 2008 (link), 2009 (link)). Collected tissues were sectioned at 6μm thickness. Immunohistochemistry was performed using ABC kit (Vectorstain) with DAB and hydrogen peroxide as chromogens, as described (Olaopa et al., 2011 (link)). The following primary antibodies were used: phospho-SMAD1/5/8 (1:40000, Cell Signaling), α-Smooth muscle actin (1:5000, Sigma), MF20 (1:100, Developmental Studies Hybridoma Bank) and PECAM-1 (1:200, BD Biosciences Pharmingen). Both MF20 and phospho-SMAD1/5/8 antibodies required 10min antigen retrieval (DAKO). Antibody diluent (DAKO), without primary antibody, was used for negative controls. For each assay, whole embryos and/or serial sections were examined for at least three individual embryos of each genotype at each stage of development. Wildtype littermates and Nkx2.5^Cre only embryos were always used as age-matched control samples.