Journal: Life science alliance

Article Title: BACH family members regulate angiogenesis and lymphangiogenesis by modulating VEGFC expression.

doi: 10.26508/lsa.202000666

Figure Lengend Snippet: Figure 5. BACH1 promotes angiogenesis and lymphangiogenesis during ovarian tumor progression in mouse models. Ex vivo analysis of subcutaneous xenografts and diaphragm specimens excised from CD-1 nude female mice implanted with control (Control) or BACH1 ectopically expressing (BACH1) human ovarian clear cell carcinoma ES2 cells. (A) Immunofluorescence labeling of blood vessels using anti-CD34 antibodies in diaphragm specimens excised from mice injected intraperitoneally with control or BACH1-expressing human ES2 cells. Scale bar, 100 μm. (B) Morphometric analysis of the diaphragm relative region covered by CD34+ blood vessels. Diaphragms were excised from mice inoculated intraperitoneally with either control (Control, n = 3) or BACH1-overexpressing (BACH1, n = 7) ES2 cells (mean ± SEM; *P = 0.0304). (C) Confocal z-projection images (Z dimension 7 μm) of control and BACH1-overexpressing subcutaneous-ES2 ovarian carcinoma xenografts subjected to LYVE1 immunofluorescence staining along with a modified CLARITY technique. Images demonstrate the complexity of the lymphatic vasculature. Scale bar, 100 μm. 3D reconstructions of the stacks are available in Videos 1 and 2. (D) Lymphatic vessel immunostaining, using anti- LYVE1 antibodies, of diaphragm specimens excised from mice injected intraperitoneally with control or BACH1-overexpressing ES2 ovarian carcinoma cells. A black arrow indicates infiltration of cells into the lymphatic vessel and an asterisk, their absence. Scale bar, 100 μm. (E) Morphometric analysis of the diaphragm relative region covered by LYVE1+ lymph vessels. Diaphragms were excised from mice inoculated intraperitoneally with either control (Control, n = 6) or BACH1-overexpressing (BACH1, n = 9) ES2 cells (mean ± SEM; *P = 0.0047). (F) Immunofluorescence double staining of LYVE1+

Article Snippet: Human ovarian clear cell carcinoma ES2 cell line (American Type Culture Collection) was cultured in DMEM supplemented with 10% FBS.

Techniques: Ex Vivo, Control, Expressing, Labeling, Injection, Staining, Immunostaining, Double Staining

Journal: Life science alliance

Article Title: BACH family members regulate angiogenesis and lymphangiogenesis by modulating VEGFC expression.

doi: 10.26508/lsa.202000666

Figure Lengend Snippet: Figure 6. BACH1 and VEGFC genetically interact. (A) Conservation of BACH sites in human, mouse, and zebrafish. The distal site is completely conserved. The proximal site is fully conserved between mouse and human, whereas there are three BACH sites at very close proximity in zebrafish. All proximal sites differ by one nucleotide from the consensus sequence. (B) Chromatin immunoprecipitation assay, followed by PCR measurements, was performed using primer mapping to the above human BACH proximal and distal regulatory sites and DNA precipitated with nonspecific IgG, HA-tag, or BACH1 antibodies. (C) Schematic representation of the wild-type human VEGFC promoter-driven luciferase (Luc) reporters (blue) (pVEGFCwt-Luc) and of three constructs deleted either from proximal (nt. −623 to −603, pVEGFCΔPro-Luc) or distal (nt. −2074 to −2054, pVEGFCΔDis-Luc) BACH- binding sites or a combination thereof (nt −623 to −603 and −2074 to −2054, pVEGFCΔProDis-Luc). Numbers refer to the nucleotide positions relative to ATG (translation initiation). (D) Quantification of dual–luciferase activity in human ES2 cells driven from pVEGFCwt-Luc, pVEGFCΔPro-Luc, pVEGFCΔDis-Luc, and pVEGFCΔProDis-Luc constructs. Relative luciferase activity is shown as a percentage of the pVEGFCwt-Luc value (mean ± SEM, n = 3). *P < 0.0001, Kruskal–Wallis test. (E) Immunofluorescence staining of human ES2 cells stably expressing either an empty pIRES vector (Control) or N-terminally HA-tagged BACH1 (BACH1) with antibodies directed against the HA tag (red, left panel) or against VEGFC (red, right panel). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. TSS, transcription start site.

Article Snippet: Human ovarian clear cell carcinoma ES2 cell line (American Type Culture Collection) was cultured in DMEM supplemented with 10% FBS.

Techniques: Sequencing, Chromatin Immunoprecipitation, Luciferase, Construct, Binding Assay, Activity Assay, Staining, Stable Transfection, Expressing, Plasmid Preparation, Control