Fig 1: Immortalized BTCs maintain the epithelial-origin and endocrine features of primary BTCs without exhibiting features of malignant transformation. A) The endocrine ability of PL was measured by ELISA. B) The migration ability of BTCs was measured by trans-well chamber migration assay; bars = 50 µm. C) CK7, CD90, vimentin, and E-cadherin expression levels were detected by Western blot analysis. Lane 1, primary BTCs; lane 2, hTERT-BTCs at Passage #30; lane 3, hTERT-BTCs at Passage #50; lane 4, fetal bovine fibroblast cells (FBFs) as the negative control. D) The protein expression levels were measured using ImageJ in the representative Western blot analysis. E) hTERT-BTCs did not form cell colonies while HeLa cells formed colonies larger than 100 mm in diameter after a 14-day culture. The results are presented as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01 compared with primary BTCs).
Fig 2: Identification and hTERT-transfection of primary bovine trophoblast cells (BTCs) derived from bovine placenta. A, B) Morphological characteristics and Giemsa stain results of BTCs under a phase-contrast inverted microscope; bar = 1 mm in Fig. 1A, bar = 100 µm in Fig. 1B. C) Cytokeratin 7 (CK7), vimentin, E-cadherin, and CD90 expression in primary BTCs were evaluated by immunocytochemistry. The BTCs incubated with PBS instead of primary antibody and fetal bovine fibroblast cells served as the negative controls; bars = 100 µm. D) The growth and migration features of BTCs were analyzed by CCK-8. E, F) hTERT expression was detected by Western blot analysis. Lane 1, primary BTCs; lane 2, hTERT-BTCs at Passage #30; lane 3, hTERT-BTCs at Passage #50; lane 4, HeLa cells as positive control. G) Representative mRNA expression levels were measured by qRT-PCR and analyzed using the 2–??Ct method. The results are presented as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01 compared with primary BTCs).
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