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Blood, Vol. 112, Issue 5, 1904-1911, September 1, 2008
Dll4 activation of Notch signaling reduces tumor vascularity and inhibits tumor growth
Blood Segarra et al.
112: 1904
Supplemental materials for: Segarra et al
Files in this Data Supplement:
- Figure S1. Expression of Notch1 and Notch4 is undetectable in BL41 cells (JPG, 75.8 KB)
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Failure to detect Notch1 or Notch 4 mRNA (A) and protein (B) from parental, vector- and Dll4-transduced BL41. RT-PCR was used to detect mRNA; and immunoblotting with antibodies to Notch1 and Notch4 intracellular domain (NICD) was used to detect protein. Resting HUVEC served as control for RT-PCR; activated HUVEC served as control for immunoblotting.
- Figure S2. Dll4 inhibits VEGF-induced endothelial cell proliferation and VEGFR2 expression (JPG, 105 KB)
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HUVEC were cultured in microwells coated with recombinant Dll4-Fc with or without VEGF (25 ng/ml): (A) proliferation at 72hr was measured by uptake of radioactive thymidine (results reflect the means +/− SD of triplicate cultures), and (B) VEGFR2 mRNA was measured after 48hr by real-time RT-PCR (the bars reflect the relative group means). HUVEC were incubated (48hr) alone or co-cultured with control BL41 (parental or Vector-transduced) cells) or Dll4-BL41: (C) VEGFR2 mRNA was measured by real-time RT-PCR (the bars reflect the relative group means), and (D) VEGFR2 protein was measured by immunoblotting the cell lysates.
- Figure S3. Overexpression and function of human Dll4 in the murine MOPC315 plasmacytoma cell line (JPG, 165 KB)
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Human Dll4 transduction in the murine MOPC315 myeloma cells induced increased expression of human Dll4 mRNA (A) and protein (B–C), without affecting MOPC315 cell proliferation (D). Dll4-MOPC315 cells induced Notch activation (E) and HEY1/HEY2 expression (F) in the co-cultured HUVEC (after removal of MOPC315 cells). Reduced VEGF (25 ng/ml)-induced proliferation (G) in HUVEC monolayers after co-culture with Dll4-MOPC315 as opposed to control-MOPC315 cells. Bars represent the mean +/− s.e.m. (n=3).
- Figure S4. MOPC315 cells express a constitutively active Notch1 receptor (JPG, 73.7 KB)
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Amplification of murine Notch1 mRNA by RT-PCR from MOPC315 parental, vector- and human Dll4-transduced MOPC315 cells. There was no amplification of murine Notch4 mRNA (A). Activated Notch1 detected by immunoblotting with antibodies to Notch1 intracellular domain (NICD) in MOPC315 cells (B).
- Figure S5. Notch is activated in the tumor microenvironment of MOPC315 tumors overexpressing human Dll4. Human Dll4 mRNA detected by real-time PCR (JPG, 143 KB)
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(A) and protein (pink) detected by immunohistochemistry (B). NICD1 and NICD4 were detected by immunoblotting with human/mouse specific antibodies (C). Mouse HEY2 mRNA levels measured by real-time PCR (mouse-specific probes) in tumor tissues of Dll4 and control MOPC315 tumors (D). Bars represent the relative mean +/− s.e.m (n=13 a, d) mRNA levels.
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