Fig. 5: ML323 treatment prevents USP1 autocleavage and sensitizes cells to DNA replication and growth defects induced by SPRTN depletion.

HCT116 cell lines were treated with or without 2.5 μM ML323 inhibitor for 6 h and subjected to either immunoblotting analysis in (a) or DNA fiber analysis of elongating fork speed (b). Data are graphed with mean and −/+ SD for 3 independent experiments (n = 200 elongating forks), and p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, **p < 0.01, ****p < 0.0001, two-tailed). c, d HCT116 cells were treated with siRNAs for siCtrl or siSPRTN-1 and transfected with the indicated siRNA-resistant Flag-SPRTN constructs. Cells were then treated with or without 2.5 μM ML323 and subjected to immunoblotting analysis in (c) or DNA fiber analysis for elongating fork speed (d). CldU tract lengths are plotted for three independent experiments (n = 200 elongating forks) with mean −/+ SD indicated and p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, ****p < 0.0001, two-tailed). e HCT116 cells were transfected with either siCtrl or siSPRTN-1 siRNAs and treated with or without 2.5 μM ML323 inhibitor. Cell population doublings were calculated at 3 and 7 days for three independent experiments, with mean −/+ SD indicated. f Cytotoxicity assay of control or SPRTN-depleted HCT116 cells, treated with either cisplatin alone or a combination of cisplatin and ML323 at a ratio of 1:4. The data represent the mean ± SD of three independent experiments.