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. 2024 Jan 12;15(1):40. doi: 10.1038/s41419-023-06417-4

Fig. 5. Cholesterol homoeostasis is an important resistance mechanism for TRAIL-induced apoptosis.

Fig. 5

A Caspase-8 activity and (B) Caspase-3/-7 activity was measured following treatment with increasing concentrations of izTRAIL (0–50 ng/mL). Activity was measured 6 h post treatment using the Caspase-3/-7 or the Caspase-8 Glo assays. Cells were treated with U18666A (1 ng/ml) for 24 h. C Western blot analysis of cell lysates after treatment with U18666A and izTRAIL for key apoptotic proteins; caspase-8, -3, Bid and PARP. D–G Quantification of an inhibition of apoptosis in OE33 cells treated with U18666A (1 ng/ml) in combination with izTRAIL, showing a significant loss of cleavage of caspase-8, -3, Bid, and PARP in three independent experiments. H Bar graph showing the mean fluorescence of cell surface expression of TRAIL-R2 stained control and cells treated with U18666A in three independent experiments. I Bar graph showing the mean fluorescence intensity of cell surface expression of TRAIL-R1 in control and U18666A-treated (1 ng/ml) OE33 cells in three independent experiments (n = 10,000 cells per experiment). J Fluorescence images of OE33 cells showing the accumulation of cholesterol, stained with Filipin-III, in mitochondria after U18666A treatment. Scale bar, 5 µm. Statistical significance was measured by Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.