Figure 3.

Ugt1 knockout in intestinal crypt stem cells reduces p53 activation by etoposide. Intestinal crypt stem cells were cultured from Ugt1^F/F and Ugt1^ΔIEC adult mice. After passage, approximately 500–1000 cells per 50 μl of Matrigel per well were cultured in 24-well plates. Four days later, cells were exposed to fresh medium containing vehicle control or different concentrations of etoposide (ETO). Twenty four hours after ETO exposure, cells were collected in RIPA buffer. Whole cell extracts were prepared and protein concentrations were quantitated. Thirty μg of protein were used for gel electrophoresis and Western blot analysis. Primary antibodies directed towards UGT1A proteins (Santa Cruz Biotechnology), phosphorylated P53 (Ser 15, Cell Signaling Technology), p21 (Cell Signaling Technology), and active Caspase 3 (Cell Signaling Technology) were used. Shown is a representation of multiple Western blots. Quantitation of multiple Western blots was performed by density analysis visualized by a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Each band was normalized to α-tubulin and expressed as “Fold of control”. Since induction patterns were being compared between two groups, the Ugt1^F/F and Ugt1^ΔIEC mice, statistical comparisons were performed using two-way Anova followed by the Benferroni correction using Graphpad Prism. *P < .05.