Fig 1: Tumor angiogenesis parameters measured in young and aged OVX mice bearing 4T1 or 67NR mouse mammary gland cancer. (A–C) contrast-enhanced ultrasonography (CEUS) analysis of blood flow after contrast agent injection in (A) 4T1 bearing young mice, (B) 67NR bearing young mice, and (C) 67NR bearing aged OVX mice. N = 4–5 mice. Parameters describing blood flow in tumor tissue: PE—peak enhancement; WiR—wash-in-rate; TTP—time to peak, WiAUC—wash-in area under the curve. (D) Immunohistochemical examination of tumor CD31 expression in young mice bearing 4T1 and 67NR tumors. N = 4–5 mice. (E) Vascular endothelial growth factor (VEGF) plasma level in young mice bearing 4T1 and 67NR tumors; ELISA assay. N = 9–10 mice. (F) Representative pictures of WIR parameter measured in young and aged ovariectomized mice bearing 67NR tumors. (G) Representative images of CD31 tumor tissue staining. Scale bars = 50 µm. Statistical analysis: (A–C and E) Sidak’s, (D) Dunnett’s multiple comparison tests; *p < 0.05; **p < 0.01.
Fig 2: Characteristic of Th17 lymphocytes ex vivo differentiated with CD29, CD51, and CD44 blockade. Percentage of (A) all IL-17+, (B) IL-17+IFNγ−, (C) all IFNγ+, (D) IL-17−IFNγ+, (E) double positive IL-17+IFNγ+ cells among CD3+CD4+ splenocytes differentiated toward Th17 in control conditions (left part of each graph) and with blocking antibodies (right part of each graph). Left column of graphs: CD29 blocking, middle column: CD51 blocking, and right column, CD44 blocking. CD3+CD4+ splenocytes isolated from 4T1 young mice treated with calcitriol and tacalcitol (blocked or not with antiCD29, antiCD51, and antiCD44 antibodies before seeding) were differentiated ex vivo on plates coated with antiCD3 and antiCD28 antibodies with the presence of IL-6 and TGF-β. On the last day, cells were incubated with PMA (50 ng/mL) + ionomycin (500 ng/mL) + brefeldin A (5 µg/mL) and incubated for 4 h. The cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFNγ. N = 4–5. Statistical analysis: Sidak’s multiple comparisons test; *p<0.05.
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