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. 2016 Dec 8;16(4):348–359. doi: 10.1080/15384101.2016.1260210

Figure 4.

Figure 4.

NOX4 activated RIPK1-related NF-κB signaling pathways in cardiac hypertrophy. (A) Western blotting analysis for RIPK1 and phosphorylated P65 subunit of NF-κB in rats 4 weeks after TAC or sham surgery. n = 6. * indicates P<0.05 vs. sham-operated group. (B) Western blotting analysis for RIPK1 and phosphorylated P65 subunit of NF-κB in NCMs treated with AngII (10−5M for 24h, Top) or ISO (10−5M for 24h, Bottom). n = 4. * indicates P<0.05 vs. control. (C) Western blotting analysis for RIPK1 and phosphorylated P65 subunit of NF-κB in NCMs in the presence or absence of AngII (10−5M for 24h, Top) or ISO (10−5M for 24h, Bottom) after transfected with si-Con or siNOX4. n = 4. * indicates P<0.05 vs. si-Con, # indicates P<0.05 vs. si-Con+AngII or si-Con+ISO. (D) Western blotting analysis for RIPK1 related NF-κB signaling in NCMs. NCMs treated with AngII (10−5M for 24h), ISO (10−5M for 24h) or H2O2 (100μM for 2h) showed significantly higher activation of RIPK1/NF-κB signaling compared to blank group. The activation of RIPK1/NF-κB signaling was restored by pretreatment with either NAC (10mM for 1h) or GKT137831(5μM for 1h). n = 3. *, ## indicates P<0.05 vs. blank and DMSO group, respectively; ** indicates P<0.05(AngII, ISO and H2O2 group vs. AngII+NAC, ISO+NAC and H2O2+NAC group, respectively); # indicates P<0.05(AngII and ISO vs. AngII+GKT137831 and ISO+ GKT137831 group, respectively). (E) Western blotting analysis for RIPK1 and phosphorylated P65 subunit of NF-κB in NCMs in the presence or absence of AngII (10−5M for 24h, Top) or ISO (10−5M for 24h, Bottom) after transfected with si-Con or siRIPK1. n = 4. *, ** indicates P<0.05 vs. si-Con, # indicates P<0.05 vs. si-Con+AngII or si-Con+ISO. (F) Immunoprecipitation analysis for K63-linked polyubiquitination of RIPK1. RIPK1 immunoprecipitates from whole-cell lysates of NCMs were subjected to Western blotting. NCMs treated with AngII (10−5M for 24h) or ISO (10−5M for 24h) showed significantly higher K63-linked polyubiquitination of RIPK1 as compared to control group. The polyubiquitination of RIPK1 was restored by transfection of si-Con or siNOX4 into NCMs treated with AngII (10−5M for 24h) or ISO (10−5M for 24h). (G) Western blotting analysis for RIPK1 and phosphorylated P65 subunit of NF-κB in NCMs treated with AngII (10−5M for24h, Top) or ISO (10−5M for 24h, Bottom) after pretreatment of Nec-1(10nM). n = 4. * indicates P<0.05 vs. control. (H) Surface area determination of NCMs in the presence or absence of AngII(10−5M for 24h) or ISO (10−5M for 24h) after transfected with si-Con or siRIPK1. n = 4. The fluorescent micrograph is representative of cells from 4 independent visual fields. * indicates P<0.05 vs. si-Con; # indicates P<0.05 vs. si-Con+AngII or si-Con+ISO.