Fig 2: Schematic summary of O2•-/ONOO--mediated activation of NF-?B via redox inactivation of PP2A promotes invasiveness.Downregulation or inhibition of SOD1 induces a pro-oxidant milieu favoring elevated intracellular O2•- upregulates ONOO- level through its reaction with NO. Resulting downstream ONOO- nitrates and inactivates B56?3-containing PP2A holoenzyme assembly by preventing the recruitment of PP2A AC catalytic core to IKKß. This consequentially results in accumulation of p-IKKa/ß leading to activation of NF-?B signaling which promotes tumor cell invasion and migration potential.

Fig 3: Cardiac expression of PP2A catalytic, scaffolding and regulatory subunits. Protein levels of PP2AC a isoform, PP2AC a and ß isoforms, PP2AA a and ß isoforms, B55a, B56?, B56d, and a4 in ventricular tissue from WT, HET, and HOM mice, assessed by immunoblotting. Lines show mean ± SE (n = 5–6 per group). There were no significant differences between groups (one-way ANOVA).

Fig 4: Generation and characterization of mice deficient for B56a. (A) Schematic of the gene encoding B56a (Ppp2r5a; exons in black) showing the insertion site of the gene trap cassette (BGEO) and locations of forward (F1, F2) and reverse (R) primers used to amplify regions of the WT and mutant alleles by PCR from genomic DNA of WT, HET, and HOM mice. A DNA gel illustrating the outcome of a typical genotyping run is also shown. (B) Protein levels of B56a in ventricular tissue from WT, HET, and HOM mice, assessed by immunoblotting (n = 6 per group). (C) PP2A catalytic activity in ventricular tissue from WT, HET, and HOM mice, assessed by a quantitative assay that uses 32P-labelled phosphorylase a as substrate (n = 6–8 per group). Lines show mean ± SE; *P < 0.05 (one-way ANOVA with Tukey’s post hoc test).

Fig 5: Accumulation of IKK phosphorylation is a function of redox-inactivation of B56?3-containing PP2A holoenzyme (PP2AB56?3)A. Western blot analysis showing p-IKKa/ß, pSer32/36I?Ba, IKKß, I?Ba, Snail, Bcl-xL, XIAP and tubulin following 6-h treatment with increasing concentrations of okadaic acid (10–40 nM) in U2OS cellsB. Western blot analysis showing p-IKKa/ß, IKKß, siB56a, siB56ß, siB56d, siB56?, and siB56e and tubulin 48-h after transfection with control siRNA or siB56a, siB56ß, siB56d, siB56?, and siB56e (100 nM) in U2OS cells (n = 2)C. Western blot analysis showing p-IKKa/ß, IKKß, HA-Tag and tubulin following 4-h treatment with 100 µM DDC in U2OS cells transiently transfected with empty vector pCEP4 or HA-B56?3 (2 µg) (n = 2)D. Western blot analysis showing the immunoprecipitation of IKKß and immunoblotting of B56? following 4-h treatment with 100 µM DDC in U2OS cells (n = 2)E. Western blot analysis showing the immunoprecipitation of IKKß and immunoblotting of B56? following 2-h treatment with 50 µM ONOO- in U2OS cells (n = 2)F. Western blot analysis showing the immunoprecipitation of B56? and immunoblotting of p-IKKa/ß following 4-h treatment with 100 µM DDC in U2OS cells transiently transfected with HA-B56?3 (8 µg) (n = 2)G. Proximity ligation assay (PLA) showing red foci (dots) indicating endogenous interaction between PP2A catalytic subunit (PP2Ac) and IKKß following 4-h treatment with 100 or 400 µM DDC, 6-h treatment with 50 µM FeTPPS, 2-h treatment with 50 µM ONOO- or 6-h treatment with 1 mM SNP, respectively, in U2OS cells. Random representative fields were imaged and the total number of foci in 100 random cells were counted (*** = p < 0.001)H. Western blot analysis showing the immunoprecipitation of HA-B56?3 and immunoblotting of 3-nitrotyrosine (3-NT) following 4-h treatment with 100 µM DDC in U2OS cells transiently transfected with HA-B56?3 (n = 2).