Figure 3
Knockdown expression of CnB, NFAT1 or NFAT2 impairs in vivo tumor growth. (a) Immunohistochemical analysis of NFAT1 expression and subcellular localization in control (LKO), CnB1-silenced (shCnB) and NFAT1-silenced (shNFAT1) 4T1 tumors. A lymph node section of control mice (left panel) has been used as positive control for NFAT1 expression and nuclear localization. Arrows point to NFAT1-expressing lymphoid infiltrating cells, whereas stars point to nuclear NFAT1 in mammary tumor cells. (b) 4T1 cells (5 × 105; n=4 for each 4T1 cell derivatives) were inoculated in the inguinal mammary fat pad of syngeneic Balb/c mice. Tumor size was measured every 3 days with a caliper and the tumor volume calculated according to the equation V= (L × W2)/2. Data are represented as mean±S.E.M. (c) Tumor volume at the time of killing, 3–5 weeks after inoculation. Control pLKO tumors (white dots, n=21), shCnB1 tumors (light gray, n=21), shNFAT1 tumors (black dots, n=21), shNFAT2 tumors (gray dots, n=16). Data are represented as mean±S.E.M. (d) Quantification of the number of proliferative cells (Ki67+, upper panel) and apoptotic cells (CC3+, lower panel) in tumors (n=3) 15 days after inoculation. Numbers are the results of counting five consecutive fields at the × 20 magnification. Data are represented as mean±S.E.M. (e) Tumors excised 15 days after orthotopic injection were fixed, paraffin embedded and further analyzed for the expression of cleaved caspase 3 (CC3) by immunohistochemistry. The right panels represent a higher magnification of the insets shown in respective left panels. A representative picture of each tumor is shown. Arrows point to CC3-positive cells. Note the unspecific brown staining of necrotic areas observed at the × 1 magnification
