Figure 6.
Tip60 interacts with and acetylates Rheb to regulate mTORC1 activity and insulin sensitivity under PA condition. (A) Relative mRNA levels of acetyltransferase genes (cbp, gcn5, pcaf, and tip60) were analyzed by quantitative PCR in fish myocytes under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (B) Relative mRNA levels of acetyltransferase genes (cbp, gcn5, pcaf, and tip60) were analyzed by quantitative PCR in C2C12 myotubes with control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (C) Immunoblotting of S6K phosphorylation in fish myocytes and C2C12 myotubes treated with control or 150 μM MG149 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (D) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 under control or 500 μM PA treatment (n = 3 independent wells per treatment). (E) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for co-immunoprecipitation (Co-IP) to assay the interaction between Rheb and Tip60. IP was performed with anti-Flag beads or anti-HA beads. (F) HEK293T cells were transfected with FLAG-Rheb together with or without HA-Tip60 plasmids in the presence or absence of PA, and the acetylation levels of Rheb and the phosphorylation levels of S6K were measured via immunoblotting. IP was performed with anti-Flag beads. (G) HEK293T cells were transfected with plasmids as indicated, and acetylation levels of Rheb were measured via immunoblotting. IP was performed with anti-Flag beads. (H) In vitro acetylation assay was conducted by incubating recombinant protein Rheb purified from E. coli with Tip60-HA protein immunopurified from HEK293T cells in HAT buffer at 37°C for 1 h. (I) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with control or 150 μM MG149 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (J) Immunoblotting of Rheb acetylation and S6K phosphorylation in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) Immunoblotting of Rheb acetylation in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). IP was performed with an antibody to Rheb. (L) Insulin-stimulated glucose uptake was measured by 2-DG uptake assays in fish myocytes treated with control or 150 μM MG149 in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (M) AKT phosphorylation levels were assayed by immunoblotting in fish myocytes and C2C12 myotubes (n = 3 experimental replicates). Cells were pretreated with control or 150 μM MG149 treatment in the absence or presence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (N) AKT phosphorylation levels were assayed by immunoblotting in C2C12 myotubes (n = 3 experimental replicates). Cells were transfected with control siRNA or siRNA against Tip60 and pretreated with control or 500 μM PA for 12 h, and then stimulated with 100 nM insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t tests (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available for this figure: SourceData F6.

Tip60 interacts with and acetylates Rheb to regulate mTORC1 activity and insulin sensitivity under PA condition. (A) Relative mRNA levels of acetyltransferase genes (cbp, gcn5, pcaf, and tip60) were analyzed by quantitative PCR in fish myocytes under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (B) Relative mRNA levels of acetyltransferase genes (cbp, gcn5, pcaf, and tip60) were analyzed by quantitative PCR in C2C12 myotubes with control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (C) Immunoblotting of S6K phosphorylation in fish myocytes and C2C12 myotubes treated with control or 150 μM MG149 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (D) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 under control or 500 μM PA treatment (n = 3 independent wells per treatment). (E) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for co-immunoprecipitation (Co-IP) to assay the interaction between Rheb and Tip60. IP was performed with anti-Flag beads or anti-HA beads. (F) HEK293T cells were transfected with FLAG-Rheb together with or without HA-Tip60 plasmids in the presence or absence of PA, and the acetylation levels of Rheb and the phosphorylation levels of S6K were measured via immunoblotting. IP was performed with anti-Flag beads. (G) HEK293T cells were transfected with plasmids as indicated, and acetylation levels of Rheb were measured via immunoblotting. IP was performed with anti-Flag beads. (H) In vitro acetylation assay was conducted by incubating recombinant protein Rheb purified from E. coli with Tip60-HA protein immunopurified from HEK293T cells in HAT buffer at 37°C for 1 h. (I) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with control or 150 μM MG149 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (J) Immunoblotting of Rheb acetylation and S6K phosphorylation in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) Immunoblotting of Rheb acetylation in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). IP was performed with an antibody to Rheb. (L) Insulin-stimulated glucose uptake was measured by 2-DG uptake assays in fish myocytes treated with control or 150 μM MG149 in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (M) AKT phosphorylation levels were assayed by immunoblotting in fish myocytes and C2C12 myotubes (n = 3 experimental replicates). Cells were pretreated with control or 150 μM MG149 treatment in the absence or presence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (N) AKT phosphorylation levels were assayed by immunoblotting in C2C12 myotubes (n = 3 experimental replicates). Cells were transfected with control siRNA or siRNA against Tip60 and pretreated with control or 500 μM PA for 12 h, and then stimulated with 100 nM insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t tests (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available for this figure: SourceData F6.

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