Su Y , Stolow MA .

	Figure 2. The tadpole intestinal epithelial cells but not the fibroblasts respond to T3 by undergoing programmed cell death. (A) T3-treatment of epithelial cells resulted in the formation of a nucleosome-sized DNA ladder. The epithelial cells were cultured on plastic dishes in the absence (Control) or presence (T3 treated) of 100 nM T3 for 1 d. The genomic DNA was isolated, electrophoresed on an agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The DNA bands equivalent to the lengths of the DNA in 1–4 nucleosomes were labeled on the right. (B) T3 induces dose-dependent DNA fragmentation in the tadpole intestinal epithelial cells. The epithelial cells were cultured on plastic dishes in the presence of different concentrations of T3 for 3 d and then DNA fragmentation was measured using the ELISA methods. Note that DNA fragmentation was detectible with as low as 5–10 nM of T3, similar to that in the plasma during metamorphosis (Leloup and Buscaglia, 1977), and plateaued at 100 nM T3. (C) Kinetics of T3-induced epithelial cell DNA fragmentation. The intestinal epithelial cells were cultured in the presence of 100 nM T3 for 1–5 d and then DNA fragmentation was analyzed with the ELISA method. Note that DNA fragmentation reached the maximum after 3 or 4 d of treatment. (D) T3 induces DNA fragmentation in the epithelial cells but not the fibroblasts. Intestinal epithelial cells and fibroblasts were isolated and cultured on 96-well plastic dishes (2 × 104 cells/well) for 1 or 3 d in the presence or absence of 100 nM T3. DNA fragmentation was then determined by using the ELISA method.
	Figure 3. Flow cytometry analysis indicates that epithelial cells undergo apoptosis in response to T3 at different stages of the cell cycle. The epithelial cells were cultured in the presence or absence of 100 nM T3 for 2 or 3 d. The cells were then analyzed by flow cytometry. Although the exact boundary between the live cells and apoptotic cells (encircled area) was difficult to determine with precision, the results clearly showed that cells with different DNA contents or at different cell cycle stages (G2 at the top and G1 at the bottom) were present in the apoptotic region (reflected by the increased cellular granularity). Note that after 3 d of treatment, essentially all cells were in the apoptotic region, and were shown to be dead by trypan blue staining and DNA fragmentation (Figs. 1 and 2).
	Figure 4. T3 stimulates the proliferation of both the intestinal epithelial cells and fibroblasts. Intestinal epithelial cells and fibroblasts were cultured overnight on 96-well plastic dishes (5 × 104 cells/well) in the presence or absence of 100 nM T3. 0.1 μCi of [3H]thymidine was added to the 0.1-ml culture medium/well and incubated for another 5 h. The amount of [3H]thymidine incorporated into genomic DNA was then measured.
	Figure 5. T3 treatment of intestinal epithelial cells leads to the downregulation of two known epithelial specific genes. (A) Kinetics of the downregulation of IFABP gene by T3 in vitro. Epithelial cells from stage 57/58 tadpole intestine were cultured on plastic dishes in the presence of 100 nM T3 for indicated numbers of hours and then RNA was isolated for Northern blot analysis of IFABP mRNA. (B) Regulation of IFABP and Na+/ PO43− cotransporter genes by T3 in vitro. Epithelial cells from stage 57/58 or stage 64 tadpoles were isolated and cultured on plastic dishes in the presence or absence of 100 nM T3 for 1 d. The RNA was isolated and analyzed by Northern blot hybridization with the cDNA probes for IFABP and intestinal Na+/PO43− cotransporter (NaPi). Note that both genes were downregulated in the stage 57/58 epithelial cells as expected from their expression during normal development (Shi and Hayes, 1994; Ishizuya-Oka et al., 1994, 1997). However, their downregulation in stage 64 epithelial cells appeared to contradict with expectation (see text for discussion). The hybridization with rpL8 served as a loading control.
	Figure 6. Both larval and adult intestinal cells undergo apoptosis upon T3 treatment in vitro. Cells were dissociated from intestine of stage 57/58 or 64 tadpoles and all of the cells were cultured together in vitro in the presence or absence of 100 nM T3. Cell death was analyzed by using the DNA fragmentation ELISA assay. The cells were predominantly epithelial but a higher portion of mesenchymal cells were present in stage 64 tadpole intestine (McAvoy and Dixon, 1977; Ishizuya-Oka and Shimozawa, 1987a). Note that cell death were detected for both the larval and adult intestinal cells. Slightly lower levels of T3-induced DNA fragmentation were observed at stage 64, probably reflecting the presence of a slightly higher percentage of nonepithelial cells.
	Figure 7. Intestinal epithelial cells cultured on fibronectin-coated dishes survive longer in the presence and absence of T3 than those on plastic dishes. (A) The cells were cultured on the coated dishes in the presence or absence of 100 nM T3 for 0 or 7 d and then photographed. Note that the cells attached nicely to the coated dishes in contrast to their behavior on plastic dishes, and that some cells were still present after 7 d of T3 treatment, in contrast to those on plastic dishes (Fig. 1). (B) The cells were cultured on fibronectin-coated dishes in the presence or absence of 100 nM T3 and then the live cells were counted at different days after trypsinization and trypan blue staining.
	Figure 8. Epithelial cells from stage 57/58 intestine cultured on matrix-coated dishes are more resistant to T3-induced apoptosis, but are similarly stimulated by T3 to proliferate or downregulate IFABP expression. (A) Intestinal epithelial cells were cultured on plastic dishes with or without indicated coatings for 1–3 d in the presence or absence of 100 nM T3. DNA fragmentation was then measured by the ELISA method. (B) Epithelial cells cultured on various matrix-coated dishes have a similar dose response to T3 treatment in spite of their increased resistance to T3-induced cell death. The epithelial cells were cultured on various dishes in the presence of different concentrations of T3 for 3 d and then DNA fragmentation was then determined by the ELISA method. (C) Epithelial cells were cultured overnight on various matrix-coated dishes in the presence or absence of 100 nM T3 (5 × 104 cells/well). 0.1 μCi of [3H]thymidine were added into the 1-ml culture medium/well and then incubated for another 5 h. [3H]thymidine incorporated into genomic DNA was then measured. (D) Epithelial cells were cultured on matrix-coated dishes in the presence or absence of 100 nM T3 for 1 d and then total RNA was isolated for Northern blot analysis of IFABP mRNA. The hybridization with rpL8 served as a control.
	Figure 9. T3-induced intestinal epithelial cell death but not cell proliferation can be inhibited by some but not all known inhibitors of mammalian apoptosis. (A) The epithelial cells were cultured on plastic dishes for 3 d in the presence or absence of 100 nM T3 and/or 300 ng/ml CsA, 10 ng/ml FK506 (FK), 100 μM ATA, and 50 μM Z-VAD (VAD). DNA fragmentation was then measured by the ELISA method. Note that with the exception of FK506, all inhibitors blocked T3-induced epithelial cell DNA fragmentation. None of the drugs had any effect on DNA fragmentation by itself. (B) Time course of the drug inhibition of T3-induced epithelial cell death. Note that again with the exception of FK506, all drugs inhibited cell death throughout the treatment. The concentrations of the drugs used were 600 ng/ml CsA, 10 ng/ml FK506 (FK), 100 μM ATA, and 50 μM Z-VAD (VAD), respectively. (C) CsA does not block T3-induced epithelial cell proliferation. The epithelial cells were cultured in the presence or absence of 100 nM T3 and/or 600 ng/ml CsA for 1 d. Cell proliferation was determined as in Fig. 4. (D) The downregulation of IFABP gene in vitro by T3 is resistant to CsA and FK506. Epithelial cells from stage 57/58 tadpoles were cultured on plastic dishes in the presence or absence of 100 nM T3 and/or 600 ng/ml CsA or 10 ng FK506 (FK) for 1 d. The RNA was then isolated and analyzed as above. Note that FK506 had no effect on either cell death (A and B) or IFABP downregulation. Although CsA could inhibit cell death (A and B), it failed to block T3-induced IFABP gene regulation. The hybridization with rpL8 served as a loading control.

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