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. 2018 Sep 7;19:375–387. doi: 10.1016/j.redox.2018.09.007

Fig. 4.

Fig. 4

The RIPK1/RIPK3-complex is required for curcumol to induce HSC necroptosis in vitro. (A, B) Human HSC-LX2 cells were treated with DMSO (0.02%, w/v) and/or curcumol at the indicated doses for 24 h, or curcumol at 30 μM for various hours. Western blot analyses of phosphorylated RIPK1 (Ser166), RIPK3 (Ser227), MLKL(Ser358) and corresponding total protein expression in activated HSCs treated with curcumol at different concentrations for 24 h or curcumol at 30 μM for various hours. (C, D) Human HSC-LX2 cells were treated with DMSO (0.02%, w/v) and/or curcumol at the indicated doses for 24 h. Immunofluorescence staining for RIPK1, RIPK3 in HSC-LX2 cells. Scale bar, 50 µm. (E) Human HSC-LX2 cells were pretreated with 50 μM Nec-1 for 1 h, followed by curcumol treatment for 24 h. RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in HSC-LX2 cells. Data are expressed as mean ± SD (n = 3); *P < 0.05 versus DMSO, **P < 0.01 versus DMSO, ***P < 0.001 versus DMSO.