Fig 1: Loss of USP1 leads to down-regulation of CHK1 protein levels and acquisition of CCT244747 resistance.(A) Western blot and Q-PCR analysis from WT U2OS cells following siRNA targeting c-Rel or a Non-specific siRNA control. Western blot analysis shows that knockdown of c-Rel results in a reduction in USP1, USP14 and CHK1. Actin is used as a loading control. Q-PCR data shows that USP1 and USP14 transcript levels are reduced following c-Rel knockdown, but that CHEK1 transcript levels are unaffected. Data represents mean ± SEM, each point is the mean of three independent experiments. ** P < 0.01 (Unpaired student's t-test). (B) Western blot analysis from WT U2OS cells following siRNA targeting USP1, USP14 or a Non-specific siRNA control. Data shows that CHK1 is completely lost following USP1 knockdown and partially lost following USP14 knockdown. Actin is used as a loading control. (C) Western blot analysis of WT or CCT244747 resistant U2OS cells treated with CCT244747, the USP1 inhibitor ML323, or the Proteasome inhibitor MG-132, alone or in combination. Blots were probed for CHK1, USP1, yH2AX or Actin. Inhibition of USP1/14 in WT U2OS results in the loss of CHK1. Proteasomal inhibition in the CCT244747 resistant U2OS cells results in the stabilisation of CHK1 protein. (D) Clonogenic survival in WT U20S cell lines following siRNA targeting USP1, USP14 or a Non-specific siRNA control. U2OS cells are sensitive to CCT244747 in the presence of control or USP14 siRNA, however knockdown of USP1 renders them insensitive to CCT244747 treatment. Data represents mean ± SEM, each point is the mean of three independent experiments. *** P < 0.01 (One-way ANOVA with Tukey's post-hoc test). (E) Scatter showing the response of one re-implanted Eµ-Myc tumour to ML323 in the lymphoid tumour sites. One Eµ-Myc tumour was implanted into six syngeneic recipient C57Bl/6 mice, three were treated with ML323 (10 mg/kg i.p), and three with vehicle control, for 9 days once tumours became palpable. A response was defined as a significant reduction in tumour burden (P < 0.05) using unpaired Student's t-tests. WT Eµ-Myc tumour burden was reduced by ML323 treatment in all lymphoid tissues.