Fig 2: Ionizing radiation enhances ACAT1 K181 crotonylation.A Schematic diagram of the crotonylation proteomics analysis workflow. B KEGG pathway enrichment analysis of metabolic crotonylated proteins. C Schematic illustration of differentially crotonylated proteins involved in glycolysis mapped to the glucose metabolism pathway. D, E Western blot analysis of ACAT1 crotonylation and acetylation levels in cells at 4 h after 8 Gy IR treatment. F, G Western blot analysis of ACAT1 crotonylation levels in cells at 4 h after 0, 1, 2, 4, and 8 Gy IR treatment. H, I Western blot analysis of ACAT1 crotonylation levels at 0, 1, 2, 4, 8, and 12 h following 8 Gy IR treatment. J, K Mass spectrometry-based identification and quantification of crotonylation at ACAT1 K181. L, M Western blot analysis of the IR-induced crotonylation site in ACAT1. Data are mean ± SD; n = 3 each; *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig 3: ACAT1 K181 crotonylation regulates PDHA1 acetylation.A ACAT1 activity assay detecting the effects of 8 Gy IR treatment at 4 h on ACAT1 enzymatic activity. B PDHA1 activity assay detecting the effects of 8 Gy IR treatment at 4 h on PDHA1 enzymatic activity. C, D Western blot analysis of the effects of 8 Gy IR treatment at 4 h on PDHA1 acetylation, phosphorylation, and interaction with ACAT1. E, F Western blot analysis of the time-dependent effects of 8 Gy IR on PDHA1 acetylation. G ACAT1 activity assay detecting the effects of different ACAT1 sites on enzyme activity. H, I Western blot analysis of the effects of ACAT1 mutants (K181R, K181Q, K181N) on PDHA1 acetylation. J, K Western blot analysis of the role of ACAT1 K181 in IR-induced ACAT1 crotonylation. L–N Western blot analysis of the role of ACAT1 K181 in IR-induced PDHA1 acetylation and Ser293 phosphorylation. Data are mean ± SD; n = 3 each; *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig 4: Crotonylation at ACAT1 K181 modulates cellular radiosensitivity.A, B Apoptosis assays showing the effects of ionizing radiation on apoptosis in HeLa cells after ACAT1 knockout or mutation of ACAT1 at lysine 181 to arginine (n = 3). C–F Clonogenic survival assays examining the effects of ACAT1 knockout on cellular radiosensitivity in HeLa and MCF7 cells (n = 3). G, H Clonogenic survival assays showing the effects of reconstitution with ACAT1 K181R or K181N in HeLa ACAT1 knockout cells on cellular radiosensitivity (n = 3). I, J Clonogenic survival assay showing the effects of ACAT1 K181R mutation on cellular radiosensitivity in MCF7 cells (n = 3). K Tumor size in each group was monitored every 3 days beginning immediately after radiation therapy (n = 8). L, M Tumor images and tumor weight of each group at 21 days post-radiation treatment (n = 8). Data are mean ± SD; *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig 5: GCN5 mediates IR-induced ACAT1 crotonylation.A Western blot analysis of the effects of different crotonylating enzymes on ACAT1 crotonylation. B, C Western blot analysis of the dose-dependent effects of GCN5 overexpression on ACAT1 crotonylation (n = 3). D, E Western blot analysis of the effects of GCN5 knockdown on IR-induced ACAT1 crotonylation (n = 3). F Western blot analysis of the effects of IR on the interaction between ACAT1 and GCN5. G, H PLA detecting the effects of IR on the interaction between ACAT1 and GCN5 (n = 96). I, J Western blot analysis of GCN5-mediated ACAT1 crotonylation in an in vitro crotonylation assay (n = 3). K, L Western blot analysis of the effects of GCN5 and GCN5 E575Q on the crotonylation of ACAT1 and ACAT1 K181R. Data are mean ± SD; *P < 0.05; **P < 0.01; and ***P < 0.001.