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. 2021 Feb 18;13(2):315. doi: 10.3390/v13020315

Figure 4.

Figure 4

Increased caspase-3 activity and the proteolytic cleavage of Beclin-1 in ABCE1 knockdown cells on RNase L activation by 2-5A. (A) Immunoblot analysis of HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 µM of 2-5A for indicated times and probed with cleaved PARP and cleaved caspase-3 antibodies and normalized to β-actin levels. (B) The band intensity was calculated using Image J software and the ratio of cleaved PARP/β-actin or cleaved caspase-3/β-actin was determined and the levels were compared to WT cells. (C) Caspase-3/7 enzyme activity was measured in HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 µM of 2-5A for indicated times and the cleavage of fluorescent caspase-3 substrate was determined. Results shown represent mean ± SD for the experiment performed in triplicate and representative of three independent experiments. (D) Immunoblot analysis of HT1080 WT, ABCE1 KD, RNase L KO, and ABCE1 KD/RNase L KO cells transfected with 10 µM of 2-5A for indicated times and probed with Beclin-1 antibodies and normalized to β-actin levels. The band intensity was calculated using Image J software and the ratio of cleaved Beclin-1/β-actin was determined and the levels were compared to WT cells. Results are representative of three independent experiments. WT: wild-type; ** p < 0.01; *** p < 0.001; ns: not significant.