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. 2008 Mar 20;149(7):3321–3329. doi: 10.1210/en.2008-0080

Figure 4.

Figure 4

Transfection of cultured TECs of FLIP Tg+ mice with FLIP siRNA abolishes the protective effect of FLIP on Fas-mediated apoptosis 60–70% confluent cultured TECs of FLIP Tg+ mice were transfected with 10 pmol murine FLIP siRNA (right columns) or control siRNA (left columns). Three days after transfection, TECs were cultured for 4 d with 100 IU/ml IFN-γ and 50 IU/ml TNF-α and then cultured overnight with 1 μg/ml anti-Fas (the time corresponds to 8 d after transfection). Shown are IHC and TUNEL staining 8 d after transfection with control siRNA and FLIP siRNA. FLIP and FLAG staining is shown (A–D), and TUNEL staining is shown (E and F). IHC of activecaspase-8 and active caspase-3 is shown (G–J). FLIP+, FLAG+, TUNEL+, active caspsase-8+, or active caspsase-3+ cells (red) in five to six randomly selected high-power fields of three wells/group were manually counted, and the results are summarized (K, bars a–j correspond to A–J). A significant difference in the percentage of positive cells between Tg− and Tg+ TEC is indicated by the asterisk (P < 0.05). Shown are representative areas on slides. Original magnification, A–H, ×400.