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. 2017 Aug 24;8(47):83195–83206. doi: 10.18632/oncotarget.20541

Figure 4. Combined treatment with sorafenib and kahweol overcomes c-FLIP overexpression-mediated resistance.

Figure 4

(A) Caki cells were treated with 20 μM kahweol and 5 μM sorafenib for the indicated time periods. The protein (upper panel) and mRNA (lower panel) expression levels of c-FLIP and actin were determined by Western blotting and RT-PCR, respectively. (B) Caki cells were treated with or without 20 μM kahweol and 5 μM sorafenib in the presence of 20 μg/ml cyclohexamide (CHX) for the indicated time periods. The protein expression levels of c-FLIP and actin were determined by Western blotting. The band intensity of the c-FLIP protein was measured using ImageJ (public domain JAVA image-processing program ImageJ (http://rsb.info.nih.gov/ij). (C) Caki cells were pretreated with 1 μM MG132 and 5 μM lactacystin for 30 min and were then combined with 20 μM kahweol and 5 μM sorafenib for 24 h. The protein expression levels of c-FLIP and actin were determined by Western blot. (D) Vector cells (Caki/vector) and c-FLIP-overexpressed cells (Caki/c-FLIP) were treated with 20 μM kahweol (Kah) in the presence or absence of 5 μM sorafenib (sora) for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, c-FLIP and actin were determined by Western blot. (E) Vector cells (Caki/vector) and c-FLIP-overexpressed cells (Caki/c-FLIP) were treated with 500 ng/ml anti-Fas for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, c-FLIP and actin were determined by Western blot. The level of actin was used as a loading control. The values in D and E represent the mean ± SD from three independent samples. # p < 0.01 compared to the anti-Fas-treated Caki/Vec.