Recombinant human dll4

1-Oleoyl LPA was purchased from Avanti Polar Lipids Inc. Stealth siRNAs against human LPP3, LPA₆, Gα₁₃ and RhoA were purchased from Invitrogen. DAPT, thrombin, forskolin, Y27632 and anti-Flag M2 antibody were purchased from Sigma. Alexa-Fluor-594–phalloidin was purchased from Molecular Probes. Recombinant human Dll4 was from R&D Systems.

Primary human umbilical vein endothelial cells (HUVEC) were obtained from the Yale core facility and only used up to passage 8. The cells were cultured in M199 with 20% FBS (Gibco, Gemini), 100 µg/mL Heparin (Sigma), 50 µg/mL endothelial cell growth supplement (Sigma), and 1× penicillin/streptomycin (Sigma); some received 10 μM DAPT (Sigma) or DMSO, or were seeded onto surfaces pre-treated with 10 μg/mL recombinant human Dll4 (R&D). For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si-GJA4 (Dharmacon L-011669-00), si-CDKN1B (Dharmacon L-040178-00), si-NOTCH1 (Dharmacon L-007771-00), si-NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti-Gja4)^{33 (link)}, human CDKN1B (lenti-CDKN1B, cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma). In MEK1/2 inhibitor studies, cells were washed with fresh EGM-2 media overnight, and then treated for 1 h with 20 µM U0126 (Sigma) or DMSO. In cell cycle studies, cells were treated with 24 h 10 μM clotrimazole (Sigma), 8 h 2 µM palbociclib (Sigma), or DMSO for the equivalent period. Cell cultures were routinely tested for mycoplasma contamination, and no positive test was obtained over the course of these studies.

Serial dilutions of recombinant human DLL4 (R&D Systems, Minneapolis, MN, USA) ranging between 0.005 µg/mL and 5 µg/mL were prepared in PBS and used to coat multi-well plates by incubation overnight at 4 °C. Negative control wells were incubated with PBS. After removal of solution from each well, 3 × 10⁵ H1975, HCC827 and LC31 parental cells were plated and allowed to bind to coated wells for 72 h at 37 °C in 5% CO₂. Cells were then washed with ice cold PBS and lysed with RIPA buffer. Whole cell lysates were subjected to SDS-PAGE and western blotting analysis to detect levels of EGFR, Notch1, p53, Sp1, Hes1, and RBPSUH in control and Notch agonist-stimulated cells.

The full–length human DLL4 (SC113239) and its corresponding empty vector PCMV6–XL6 were purchased from the OriGene Company (USA). Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's instructions. For siRNA transfection (Sequences see Table S1), the quantity per 6–well plate was 100 pmol siRNA and 5 μL of reagent. The transfection efficiency was evaluated by Western blot analysis (Figure S1). Recombinant human DLL4 was purchased from R&D Systems (USA), dissolved in phosphate buffered saline (PBS) and coated overnight onto tissue culture dishes at 1 μg/mL in 0.2% gelatin.