Fig 2: (A) Structure of acetyl-lysine 3,5-dinitrobenzyl ester (AcK-DBE) and thioacetyl-lysine 3,5-dinitrobenzyl ester (ThioAcK-DBE). (B) Western Blots of single or dual ncAAs (AcK, ThioAcK) residues into H4 variants (H4K16_ThioAck/H4K91_AcK) with site-specific anti-acetyl histone antibodies (Abcam, ab109463, ab4627). 10 µL of the corresponding PURE reaction solution was loaded into each lane.

Fig 3: Hypoacetylation of H4K16 is a hallmark of CRC tissues. (A) Normal, primary and metastatic CRC tissues were subjected to immunohistochemical analyses using H4K16ac antibodies (Abcam, catalogue number ab109463) and a Vectastain Elite kit was used for secondary antibody and Avidin-Biotin Complex (ABC) conjugation. Staining was considered positive when a brown nuclear reaction was observed. A representative image from each condition is shown. Control; IHC was conducted without the incubation of primary antibodies. (B). IHC staining was quantified using ImageJ software (version 1.49, NIH, USA) and presented as average ± SEM. A paired Student's t-test was used to determine the statistical significance between normal, primary and metastatic carcinoma. Asterisks denote P < 0.05. CRC tissues with the indicated grades were subjected to IHC analyses as described above and a representative image is shown (C) and % positive H4K16ac was quantified and presented as average ± SEM (D). (E) A model for chromatin acetylation driven irinotecan resistance. In normal irinotecan-responsive cells, appropriate acetylation dynamics of H4K16 results in normal recruitment and retention of 53BP1, resulting in increased fork stalling and DSB persistence, ultimately causing cell death. Failure to maintain the appropriate dynamics of H4K16 acetylation results in increased accumulation of 53BP1, reduced fork stalling and ultimately cell survival. Treatment with histone deacetylase inhibitors can selectively sensitise irinotecan resistant cells, thereby overcoming drug resistance.