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. 2015 Apr 10;5:8566. doi: 10.1038/srep08566

Figure 1. PUMA deficiency protects crypt culture and Lgr5+ cells against radiation by blocking apoptosis.

Figure 1

Small intestinal crypts from WT and PUMA KO mice were plated in Matrigel and subjected to 5 Gy, or mock (Un) irradiation, 24 h after plating and harvested at the indicated time points. (A) Representative pictures of enteroids at day 6 and quantitation of enteroids with 5 or more buds. (B) Apoptosis was assessed by TUNEL staining at 24 h in WT and PUMA KO crypts. Left, representative pictures of TUNEL (green) staining. Right, quantitation of TUNEL index. (C) Representative pictures and quantitation of p-H2AX staining in cultured crypts at 4 and 24 h. (D) Representative pictures and quantitation of Ki 67 staining in cultured crypts at 24 h. (E) Expression of PUMA and p21 transcripts was analyzed by real-time RT-PCR. (F) Crypts were isolated from Lgr5-EGFP Cre-ER background. Lgr5+ apoptosis was assessed by TUNEL/GFP staining at 24 h. Left, representative pictures of TUNEL (green), Lgr5 (red) and nuclei (DAPI) staining. Right, the percentage of Lgr5+ crypts containing one or more TUNEL+ cells. (G) Expression of Olfm4, Lgr5, Bmi1 and Hopx transcripts was analyzed by real-time RT-PCR. Values are means ± SD, n = 3 wells from three different mice with each genotype. ***P < 0.001, **P < 0.01. Scale bar, A, 100 μm; B, C, D & F, 20 μm.