Journal: Science Advances

Article Title: AKR1B10 dictates c-Myc stability to suppress colorectal cancer metastasis via PP2A nitration

doi: 10.1126/sciadv.adv6937

Figure Lengend Snippet: Fig. 6. AKR1B10K125L impedes PP2A assembly to drive CRC metastasis. (A) Schematic representation of AKR1B10 truncations and Co-IP analysis of Flag-tagged AKR1B10 truncations in HCT116 cells, as indicated. FL, full length. (B) Computational docking model predicting the interaction between AKR1B10 (red) and PP2A (purple) using the Global RAnge Molecular Matching (GRAMM) algorithm. (C) Co-IP analysis showing the interaction between PPP2CA with AKR1B10WT, AKR1B10K125L, AKR1B- 10P219S, and AKR1B10K263R in HCT116 cells, using an anti-HA antibody. (D and E) Recombinant GST-tagged AKR1B10 (WT or K125L) was incubated with His-PPP2CA (D) or His-PPP2R1A (E). Bound proteins were analyzed by Western blot. Data are representative of three independent experiments. (F) Representative FACS images and quanti- fication of intracellular ROS levels in HCT116 shNC and shAKR1B10 cells expressing mock, AKR1B10WT, AKR1B10K125L, and AKR1B10C299S. (G) Alignment of K125 and C299 amino acid residues in AKR1B10 across multiple species. H. sapiens, Homo sapiens; M. musculus, Mus musculus; R. norvegicus, Rattus norvegicus; P. troglodytes, Pan troglodytes; M. mulatta, Macaca mulatta. (H) Co-IP analysis of PPP2R5A demonstrating increased 3-NT and reduced PPP2CA interaction in AKR1B10K125L and AKR1B10C299S overexpressing HCT116-shAKR1B10 cells. (I) Western blot analysis of c-Myc, phospho-Myc (p-Myc), and ITGB8 protein expression in HCT116 shNC and shAKR1B10 cells expressing mock, AKR1B10WT, AKR1B10K125L, and AKR1B10C299S. (J and K) Representative images (left) and quantification (right) of transwell assay (J), and relative PP2A activity (K) in HCT116 shNC and shAKR1B10 cells expressing mock, AKR1B10WT, AKR1B10K125L, and AKR1B10C299S. (L to O) Representative images (L) and quantification (M) of liver metastatic nodules in nude mice following splenic injection of HCT116 shNC and shAKR1B10 cells expressing mock, AKR1B10WT, and AKR1B10K125L. Representative H&E staining and IHC images of AKR1B10, ITGB8, and c-Myc staining in liver sections (N), with quantified histoscores (O) (n = 5 mice per group). (F, J, and K) n = 3. Data in (F), (J), (K), (M), and (O) are presented as mean ± SD, with unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Then, 3 μg of recombinant His- PPP2CA (CusaBio, CSB- EP018559HUc7) or His- PPP2R1A (CusaBio, CSB- EP018562HUa0) was added to the buffer containing protein- bound glutathione beads overnight at 4°C.

Techniques: Co-Immunoprecipitation Assay, Recombinant, Incubation, Western Blot, Expressing, Transwell Assay, Activity Assay, Injection, Staining