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. 2019 Jan 22;10(2):57. doi: 10.1038/s41419-018-1283-5

Fig. 4. Depletion of eIF5B enhances TRAIL-induced apoptosis by a caspase-8/9/7-mediated pathway.

Fig. 4

a Control or eIF5B-depleted cells were pre-treated for 2 h with a vehicle control (0.5% DMSO), pan-caspase inhibitor (z-VAD-fmk; 20 µm), RIP1-K inhibitor (Nec-1; 100 µm), calpain inhibitor III (Calp. inh.; 100 nm), autophagy inhibitor (3-Methyladenine; 3MA; 5 mm), caspase-9 inhibitor (z-LEHD-fmk; 50 µm), or caspase-8 inhibitor (z-IETD-fmk; 50 µm), before being treated with TRAIL as in Fig. 1. b–h Control or eIF5B-depleted cells (without caspase inhibition) were treated 4 h in the presence or absence of 100 ng/mL TRAIL, harvested in RIPA lysis buffer, and 20 µg of total protein resolved by SDS-PAGE before performing immunoblotting. b Representative images of immunoblots probing for eIF5B, caspase-8, caspase-3, full-length (F.L.) PARP, cleaved PARP, caspase-9, caspase-7, total Bid, tBid, and β-actin (internal control). c–h Quantitation of active- versus pro-caspase-8 c, active- versus pro-caspase-3 d, cleaved PARP normalized to β-actin e, active- versus pro-caspase-9 f, active- versus pro-caspase-7 g, and total Bid normalized to β-actin h. siC, non-specific siRNA; si5B, eIF5B-specific siRNA. Data are expressed as mean ± SEM for three independent biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001