The PKCϖ polymorphic variant, 374I, shows higher kinase activity, increased p21Cip1, IL-6 secretion and β-gal staining compared with PKCη-WT. (a) Representation of PKCη's structural domains and priming phosphorylation sites. The 374I polymorphism is indicated at the ATP binding site. The 374I polymorphic variant shows higher kinase activity as compared with WT-374V in (b) autophosphorylation assays and (c) exogenous substrate phosphorylation using MARCKS and MBP as substrates. Plasmid constructs (WT-374V, 374I and pHACE control vector) were transiently expressed in COS-7 cells. Immunoprecipitation and kinase assays using PMA for activation were performed as described in Material and Methods. The heavy IgG chain shows equal loading by Ponceau staining. The results represent five different experiments. (d) PKCη and the polymorphic variant enhance senescence markers. MCF-7 cells were transfected with WT-374V, 374I and the pHACE control vector and treated with H2O2 to induce senescence, as described above. The cells were lysed and the senescence markers p21Cip1, p16INK4a and pRB expression were detected using specific antibodies. β-Actin was used as a marker for equal protein loading. Results shown are representative of three independent experiments. (e) IL-6 production is dependent on the kinase activity of PKCη. MCF-7 cells were transfected and treated with H2O2 to induce senescence as described above. IL-6 secretion levels were measured using ELISA. (f) Overexpression of both PKCη and the polymorphic variant enhances SA-β-gal staining induced by oxidative stress. MCF-7 cells were transfected and treated as above. Cells were stained for both Hoechst and SA-β-gal 96 h after oxidative stress treatment. The total cell number was determined by nuclear fluorescent staining. Error bars represent the S.D.; two-tailed, unpaired sample t-test statistical analysis is shown: *P≤0.05, **/$$P≤0.001 and ***P≤0.0001 (* represents statistical significance for each non-treated and treated clone and $ represents statistical significance between different clones)