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. 2022 Oct 3;57:102496. doi: 10.1016/j.redox.2022.102496

Fig. 6.

Fig. 6

LOXL2-dependent deacetylation of aldolase A-K13 induces metabolic reprogramming in esophageal cancer progression.

(A) Flag-tagged wild-type aldolase A, acetyl-mimetic mutant aldolase A-K13Q and non-acetylatable mutant aldolase A-K13R were expressed in KYSE510 cells. Aldolase A proteins were purified by IP with Flag antibody, and aldolase activity was determined. Cells transfected with Flag-tagged vector served as the negative control. Data represent mean ± SD (n = 3). ***P < 0.001; ns, not significant. (B) Western blotting of supernatant and cell lysate of KYSE510 cells transfected with empty vector, Flag-tagged aldolase A (WT), its acetyl-mimetic or non-acetylatable mutant, including K13Q and K13R. (C) KYSE510 cells expressing Flag-tagged wild-type aldolase A, its acetyl-mimetic mutant K13Q and non-acetylatable mutant K13R were lysed and fractionated. Vimentin is used as a marker for the CF and GAPDH for the SF. Fractions from the cells transfected with Flag-tagged empty vector are controls for the fractionation procedure. (D) Immunofluorescence analysis of the effect of different aldolase A mutants from (B) on its release from the actin cytoskeleton in KYSE510 cells. Scale bar, 10 μm. (E) Left: Western blotting of aldolase A in the control cells, cells silenced for aldolase A expression, cells transfected with empty vector, Flag-tagged aldolase A (WT), its K13Q and K13R mutants; right: MTS assays of the indicated cells. (F) Representative low and high acetylation of aldolase A-K13 by IHC analysis (left) and Kaplan-Meier curves by log-rank test (right) of aldolase A-K13ac in tissue microarrays of patients with esophageal cancer (n = 258). Scale bar, 50 mm. (G) Kaplan-Meier curves by log-rank test of LOXL2 and L2Δ13 for overall survival and disease-free survival in patients of (F). (H) Kaplan-Meier estimates of overall survival and disease-free survival of patients with esophageal cancer according to LOXL2 or L2Δ13 associated with acetylated aldolase A-K13. (I) Proposed model integrating the LOXL2/L2Δ13-activated metabolic reprogramming through deacetylating glycolytic proteins in tumorigenesis and tumor progression.