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.
Fig 2: Down-regulation of CHK1 expression in CCT244747 resistant U2OS and Eµ-Myc cells.(A) Bar graph showing the relative expression of 24 DUBs that were significantly up- or down-regulated in the Eµ-Myc/cRel-/- tumours by RNA-Seq analysis. The red bars show that both USP1 and USP14 were down-regulated by ~2-fold compared with Eµ-Myc WTs. (B) Q-PCR validation of RNA-Seq analysis. Relative USP1 and USP14 transcript levels are significantly reduced in tumours from Eµ-Myc/cRel-/- (n = 6) and Eµ-Myc/RelAT505A (n = 5) when compared with Eµ-Myc WTs (n = 5). Data represents mean ± SEM. ** P < 0.01, *** P < 0.001 (Unpaired student's t-test). Data represents mean ± SEM, each point is an individual mouse. (C) Western blot analysis of USP1, USP14 or ACTIN in snap frozen tumour extracts prepared from re-implanted Eµ-Myc, Eµ-Myc/cRel-/- and and Eµ-Myc/RelAT505A tumours mouse inguinal lymph nodes 8 h following a single dose of CCT244747. USP1 and USP14 expression is lost in Eµ-Myc/cRel-/- tumours and reduced in and Eµ-Myc/RelAT505A tumours. Please note that the Actin blot from this figure is also used in another study (Supplementary Figure S2C middle panel, [38]), where the same membrane was probed with antibodies to other proteins. (D) Q-PCR data showing relative USP1 and USP14 transcript levels are significantly reduced in in four independently derived CCT244747 resistant U20S cell lines, compared with WT U20S cells. Data represents mean ± SEM. * P < 0.05 (Unpaired student's t-test). Data represents mean ± SEM, each point is the mean of three independent experiments in each of the four cell lines. (E) Western blot analysis of USP1, USP14, CHK1, or ACTIN in extracts prepared from WT and CCT244747 resistant U20S. USP1 and USP14 expression is lost in CCT244747 resistant U20S. (F) Western blot analysis of USP1, CHK1, or ACTIN in extracts prepared from WT and SRA-737 resistant Huh-7 cells. USP1 expression is lost in SRA-737 resistant Huh-7 cells.
Supplier Page from Proteintech Group Inc for USP14 antibody