Fig 1: Serum Delta-like ligand 4 (DLL4) levels in patients with mycosis fungoides (MF) and healthy controls. (A) Serum DLL4 levels in patients with MF and healthy controls. (B) Serum DLL4 levels in patients with early MF, advanced MF, and healthy controls.
Fig 2: Delta-like ligand 4 (DLL4)-induced proliferation of cutaneous T-cell lymphoma (CTCL) cell lines. CTCL cell lines were cultured with or without DLL4 and cell count was performed after 24 or 48 h. The data are presented as the mean ± standard error of the mean in each group. *p < 0.05, **p < 0.01.
Fig 3: Analysis of gene expression in the breakpoint region of chromosome 15q15 and determination of the corresponding protein levels in the normal skin fibroblasts of Li-Fraumeni Syndrome (LFS) patients and unrelated cancer cell linesA. A schematic representation of several genes located in chromosome 15q15. B. mRNA levels of the genes located in chromosome 15q15 in LFS cell lines and breast cancer cell line, MCF7 as well as neuroblastoma cell line IMR32, compared with normal human foreskin fibroblast cell line, HS27. C. DLL4 mRNA and corresponding protein levels in LFS and other cancer cell lines displayed in B. RT-PCR (upper panel) and immunoblot (lower panel) showing decreased expression of DLL4 in LFS cell lines, MCF7 as well as IMR32, in contrast to normal human foreskin fibroblast cell line, HS27. Densitometric analyses and the results shown in panel C reflect a mean ±S.E.M. from three independent experiments, performed in triplicates. Significance: ***P < 0.001 compared with control values, determined by t-test.
Fig 4: Immunohistochemical staining for Delta-like ligand 4 (DLL4) in lesional skin of patients with mycosis fungoides (MF) and normal skin. (A–C) Immunohistochemistry for DLL4 in lesional skin of patients with MF. (D, E) Immunohistochemistry for TOX in lesional skin of patients with MF. (F) Immunohistochemistry for DLL4 in normal skin. Representative figures from 22 patients with MF and 8 healthy controls, respectively. (G) Representative control staining using isotype-matched antibody. (H) Immunohistochemical scoring of DLL4-positive endothelial cells. *p < 0.05, **p < 0.01. (Magnification: A, F, G: x200, B–E: x400).
Fig 5: Role of DNA methylation, TP53 and CTCF in regulation of DLL4 gene expressionA. silenced DLL4 by DNA methylation in MDA231 cell line is reactivated by inhibitor of DNA methylation 5'-aza-dC. I) DLL 4 expression level in MDA231 cell line is compared with HS27 and MCF7 by RT-PCR assay. II) Reactivation of silenced DLL4 gene in MDA231 by the DNA methylation inhibitor, 5-aza-dC. III) Methylation status of the DLL4 promoter in MDA231 cell lines treated with 5-aza-dC and detected by MS-PCR. IV) Phase-contrast photomicrographs showing morphologic changes in MDA231 cells treated with 2, 5µM, 10µM and 25µM of 5-aza-dC for 3 days, compared with untreated control cells maintained for 3 days. B. The effect of DNA methylation on interaction between CTCF and DLL4 promoter by ChIP assay C. Reactivation of DLL4 gene expression in LFS cell line, 3335, by CTCF and TP53 siRNA treatment. D. A schematic representation of presumable mechanism of regulation of DLL4 gene expression.
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