Figure 6
CHIR99021 protects human intestinal culture and stem cells against radiation injury.
Two independent human intestinal cultures, EL1 (A-E) and MM1 (F-G), were plated and passaged in Matrigel as described in methods. CHIR99021 (2.5 μM) was added to culture medium 24 h before 5 Gy radiation and cultures were harvested at indicated times after radiation. (A) Representative pictures of enteroids at day 10 and quantitation of enteroids ≥100 μm. (B) Apoptosis of was assessed by TUNEL staining at 24 h. Olfm4 labels putative stem cells. Left, representative pictures of TUNEL (green), Olfm4 (red) and nuclei (DAPI) staining. Middle, quantitation of TUNEL index. Right, quantitation of Olfm4+ cells containing one or more TUNEL+ cells. (C) Representative pictures (left) and quantitation of p-H2AX staining (right) 24 h after radiation. (D) Expression of PUMA and p21 transcripts was analyzed by real-time RT-PCR. (E) Expression of Olfm4, Lgr5, Ascl2 and CD44 transcripts was analyzed by real-time RT-PCR. (F) Enteroid growth of MM1 culture was analyzed at day 6. (G) The expression of PUMA and p21 transcripts in MM1 culture at 24 h was analyzed by RT-PCR. Values are means ± SD, n = 3 wells. ***P < 0.001, **P < 0.01. Scale bar, A and F, 200 μm; B and C, 30 μm.
