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. 2010 Oct 16;6(7):891–900. doi: 10.4161/auto.6.7.13038

Figure 4.

Figure 4

The clonogenic capability of Bax-/- Hct116 cells is not affected by repeated TRAIL treatments despite continuous processing of caspase-8. (A) processing of caspase-8 in response to a single (left part) or multiple (right part) TRAIL treatments. Bax-/- Hct116 cells were treated once or daily for three days with TRAIL (100 ng/ml). Expression of the various caspases was assessed by immunoblotting. Please note, caspase-3 is processed only into p20, which remains inhibited in the absence of a mitochondrial contribution. The asterisks indicate unidentified protein bands. (B) Clonogenicity of Bax-/- Hct116 cells successively treated with a daily fresh dose of TRAIL (100 ng/ml) for 3 days as compared to WT Hct116 cells treated once with TRAIL (25 ng/ml, 6 hr). Colonies that developed during a 14-day culture in a methylcellulose-based medium in 35-mm plates were counted using an inverted microscope and gridded screen. The data (means ± SE M) were derived from three replicates in one of three experiments with equivalent results. Clonogenic assays were performed 24 hr after the first (d1), second (d2) and third (d3) TRAIL treatments of Bax-/- Hct116 cells. (C) Co-localization of cleaved caspase-8 with P-100 LC3-II during TRAIL-mediated autophagy. Control and TRAIL-treated Bax-/- Hct116 cells were subcellular fractionated to obtain purified mitochondria, S-100 and P-100 fractions. S-100 is regarded as cytosolic fraction, and P-100 as light membrane fraction. While LC3-I was detected in the S-100, LC3-II and cleaved caspase-8 were detected in the P-100 fraction. The loading in this experiment was cell-related, as indicated by the expression levels of Cox IV and β-actin. The presence of β-actin in the P-100 fraction may relate to its reported association with autophagosomes,34 and it serves to demonstrate an equal loading between control P-100 (lane 4) and TRAIL-treated cell P-100 (lane 8). (D) Accumulation of processed caspase-8 fragments in MP fraction of Bax-/- Hct116 cells treated with TRAIL in the presence of E64D.