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. 2016 Aug 4;6:30816. doi: 10.1038/srep30816

Figure 4. Fas mediates activation of caspase 8 and MAPK and ceramide analogs enhance FasL-induced caspase 8 activation.

Figure 4

(A) Human colon carcinoma SW480, RKO, and HCT116 cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting using anti-active caspase 8 and anti-cleaved PARP antibodies, respectively. The membranes were stripped and re-probed with anti-β-actin antibody. The pro-caspase 8, cleaved caspase 8, cleaved PARP and β-actin are indicated at the right. The locations of molecular weight markers are indicated at the left. (B) SW480 and RKO cells were cultured in the presence of FasL (100 ng/ml), IG7 (10 μM), or both FasL and IG7 for 24 hours and total lysate was prepared from the treated cells. Total lysate was analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins. (C,D) SW480 (C) and RKO (D) cells were cultured in the presence of FasL (50 ng/ml), IG7 (10 μM) or both FasL and IG7 for 1 and 3 hours. Cytosol fractions were isolated as in A and analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins.