Figure 4.

Demonstration of zmPDC protein expression in vivo in xenografts derived from HEK293T cells transfected with the tet‐on PDC‐GFP‐V5/His construct. (a) Fluorescence in situ hybridization of paraffin‐embedded sections of HEK‐293T–derived xenografts. Representative images of sections, stained simultaneously with mouse‐specific centromeric probes (green) and human‐specific centromeric probes (red), were acquired at 40× magnification. Nuclei were stained with DAPI (blue). Scale bar = 150 μm. (b) Western blot of protein extracts prepared from xenografts derived from HEK293T cells transfected with the tet‐on PDC‐GFP‐V5/His construct. Animals were given either no doxycycline (–) or 10 mg/mL doxycycline in their drinking water for 96 h prior to preparation of the protein extract (+). (c) Representative formalin‐fixed stained sections taken from xenografts grown for ∼ 21–24 d following subcutaneous implantation of cells transfected with the tet‐on PDC‐GFP‐V5/His construct. Where indicated, the mice were given 10 mg/mL doxycycline solution in their drinking water for 96 h prior to xenograft excision. PDC expression was investigated by probing the sections with anti‐GFP and anti‐V5 antibodies. Scale bars = 150 µm. (d) GFP fluorescence overlaid on bright field images acquired from a single cohort of mice at 48 and 96 h after the beginning of doxycycline administration. Ten mg/mL of doxycycline was given in the drinking water where indicated. Images were acquired with an IVIS 200 camera and were analyzed with Living Image software (both from Perkin‐Elmer). The post‐doxycycline GFP fluorescence images are representative of experiments on five animals. The tumors were located on the backs of the animals immediately above the tail (see Supporting Information Section S6).