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. 2017 Nov 3;25(2):340–352. doi: 10.1038/cdd.2017.164

Figure 4.

Figure 4

Lack of Caspase-10 leads to TNF-α production in response to 5FU and FADD-independent apoptosis. (a) (Left) Caspase-8 activity was measured in response to 5FU in HCT.shC10 cells. (Right) Double knockdown clones, HCT.shC10.shctrl, HCT.shC10.shC8 and HCT.shC10.shFADD were tested for apoptosis in response to 5FU. Data are plotted as mean±S.E.M. (n≥3). (b) 5FU-induced apoptosis was measured in HCT.shC10 cells in the presence of TNF-α and TNFR1-blocking antibodies. IgG1 and IgG2a antibodies were used as isotype controls, respectively. Data are plotted as mean±S.E.M. (n≥3). (c) TNF-α was measured in supernatants of 5FU-treated HCT.shC10 cells. Data are plotted as mean±S.E.M. (n=3). (d) HCT.shC10 cells after 5FU treatment were analysed by western blot for IκB-α and phospho-IκB-α. (e) HCT.shC10 cells expressing IκB-SR (Ad.IκB-SR) were treated with 5FU, after which TNF-α and apoptosis were measured. Ad.EGFP served as control. Data are plotted as mean±S.E.M. (n≥3). (f) HCT116 cells silenced for both caspase-10 and caspase-8 were treated with 5FU and TNF-α was measured. Data are plotted as mean±S.E.M. (n≥5). (g) Western blot for cFLIP in HCT116 and HCT.shC10 cells after 5FU treatment. (h) EGFP, cFLIPS, cFLIPL, cFLIPp43 and cFLIPD376N (FLIPDN) were expressed in HCT116 cells, and then the cells treated with 5FU before TNF-α levels were measured. Data are plotted as mean±S.E.M. (n≥2). (i) HCT.shC10 cells were silenced for cFLIP (Ad.shFLIP). TNF-α in cell culture supernatants was then determined after 5FU stimulation. Data are plotted as mean±S.E.M. (n=2). (j) Either cFLIPL or cFLIPp43 were expressed in HCT.shC8 cells. After 5FU treatment, TNF-α was measured. Data are plotted as mean±S.E.M. (n≥3). (k) Precipitates from a caspase-8 IP were probed for cFLIP, FADD and caspase-8. The lysates were from HCT116 cells overexpressing cFLIPL and treated with 5FU. Cells overexpressing EGFP were used as controls. Input controls are shown on the left. (l) End point tumour volumes of HCT.shC10 xenografts treated with 5FU are depicted in relation to starting volumes (set to 100). (animal numbers/group: n=6/shctrl, n=3/shC10].