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. 2025 Aug 22;11(34):eadv6937. doi: 10.1126/sciadv.adv6937

Fig. 5. Targeting AKR1B10 enhances c-Myc stability by disrupting PP2A assembly.

Fig. 5.

(A) Western blot analysis of c-Myc protein expression, and its phosphorylation status in HCT116 and SW1116 (shNC&shAKR1B10) cells. (B) IP-MS analysis to identify AKR1B10-interacting kinases and phosphatases. m/z, mass/charge ratio. (C) Co-IP analysis showing the physical interaction of exogenous AKR1B10 with PPP2CA in HCT116 and SW1116 cells. (D) Physical interaction of endogenous AKR1B10 with PPP2CA was detected by Co-IP with anti-AKR1B10 antibody in HCT116 and SW1116 cells. (E) Immunofluorescent staining showing the colocalization of endogenous AKR1B10 with PPP2CA in HCT116 and SW1116 cells. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Western blot analysis of PPP2CA and PPP2R5A protein expression in HCT116 and SW1116 (shNC&shAKR1B10) cells. (G) PP2A activity assay in HCT116 and SW1116 (shNC&shAKR1B10) cells (n = 3). (H) Co-IP analysis of HA-PPP2CA in HCT116 cells coexpressing Flag-AKR1B10 with varying plasmid amounts, as indicated. (I) Co-IP analysis of PPP2R5A indicating increased 3-nitrotyrosine (3-NT) levels and reduced PPP2CA interaction in HCT116-shAKR1B10 cells. Pan-Ace, Pan Acetylation. (J and K) Co-IP analysis of PPP2R5A showing reduced 3-NT levels and increased interaction with PPP2CA in the presence of DPI [10 μM for 20 hours (J)] or FeTPPS [10 μM for 3 hours (K)] in HCT116 (shNC&shAKR1B10) cells. (L and M) Representative fluorescence-activated cell sorting (FACS) images (L) and quantification of intracellular reactive oxygen species (ROS) levels, and relative PP2A activities [(M); n = 3] in HCT116 (shNC&shAKR1B10) cells treated with DPI (10 μM for 20 hours). DMSO, dimethyl sulfoxide. (N and O) His-PPP2R5A-WT or His-PPP2R5A-Y238F plasmid was transfected into HCT116-shAKR1B10 cells with or without Flag-AKR1B10. Co-IP analysis showing reduced 3-NT levels of His-PPP2R5A-Y238F (N). Western blot demonstrating decreased total c-Myc, pS62 c-Myc, and reduced ITGB8 expression in the presence of His-PPP2R5A-Y238F (O). Data in (G) and (M) are presented as mean ± SD, with unpaired Student’s t test. **P < 0.01 and ***P < 0.001.