Supplementarytable 1.Sequence of Primers for Real-Time PCR

SupplementaryTable 1.Sequence of primers for real-time PCR

Primer / Sequence (5'to3')
Dll4 forward primer / AGGCCTGTTTTGTGACCAAG
Dll4 reverse primer / CTCCAGCTCACAGTCCACAC
Notch1 forward primer / TCCACCAGTTTGAATGGTCA
Notch1 reverse primer / AGCTCATCATCTGGGACAGG
Notch2 forward primer / ATGACTGCCCTAACCACAGG
Notch2 reverse primer / TGCAGTCATCTCCACTCCAG
Notch3 forward primer / TCTTGCTGCTGGTCATTCTC
Notch3 reverse primer / TGCCTCATCCTCTTCAGTTG
HES1 forward primer / TGAAGAAAGATAGCTCGCGG
HES1 reverse primer / GGTACTTCCCCAGCACACTT
HEY1 forward primer / TGGATCACCTGAAAATGCTG
HEY1 reverse primer / CGAAATCCCAAACTCCGATA
PTEN forward primer / TTGGCGGTGTCATAATGTCT
PTEN reverse primer / GCAGAAAGACTTGAAGGCGTA
GAPDH forward primer / GAGTCAACGGATTTGGTCGT
GAPDH reverse primer / TTGATTTTGGAGGGATCTCG

Supplementary Table 2. Information of antibodies

Name / Isotype / Company
Notch1 (mN1A) / Mouse / Biolegend Biotechnology, Inc
Notch1 (Val1744)(D3B8) / Rabbit / Cell signal Technology
Notch1 / Rabbit / Abgent Inc
CD34 / Mouse / AbD Serotec
Ki67 (sp6) / Rabbit / Thermo Scientific
PTEN / Rabbit / Protein tech Inc
p-PTEN / Rabbit / Epitomics
p-Akt / Rabbit / Cell Signal Technology
Akt / Rabbit / Cell Signal Technology
GAPDH / Mouse / Santa Cruz Biotechnology, Inc

Supplementary figure legends for the manuscript entitled “Cross-talk Between Endothelial Cells and Tumor via Delta-like ligand4/Notch/PTEN Signaling Inhibits Lung Cancer Growth”, by Ding et al.

Supplementary Fig.S1Effect of endothelial Dll4 on the proliferation and apoptosis of NSCLC cells. a, Identification of HEVEC stable transfectants by realtime-PCR and western blot. **, P<0.01.b, A549 cells were co-cultured with EC-Dll4, EC-shDll4 or EC-EVfor 48h. The cells were then stained by propidium iodide (PI) followed by FACS analysis (upper). Apoptosis ratioof A549 cells was presented.Columns, mean; Bar, SD (lower). c, The proliferation of tumor cells next to vasculature was expressed as the number of Ki67 positive cells per field.

Supplementary Fig.S2Notch1 plays a suppressive role in the proliferation of A549 cells. a. A549 cells transiently transfected with the plasmid expressingN1ICD, shNotch1 or vector control and then cultured for 96h. Crystal violet staining was performed and the proliferation of A549 cells was measured using Cell Counting Kit-8.b. Stable transfectant of A549 cells expressing shNotch1were established and subjected toreal time-PCR. c.A549 cells stably transfected with plasmid expressing shNotch1 or control wastreated with rhDll4 or BSA for 72h, andthe proliferation of the cells was assessed by CCK-8 assay.

Supplementary Fig.S3Endothelial Dll4 up-regulates PTEN expression via Notch-dependent pathway. a. A549 cells were treated with the culture medium from EC-EV, EC-Dll4 or EC-shDll4 cells for 72h and then subjected to PCR analysis. b. A549 cells were treated with rhDll4 or BSA for 72h and subjected to western blot assay. c. 293Tcells transiently tranfected with the plasmid expressing N1ICD,shNotch1 orthe controlwassubjected to westernblot analysis.