Fig. 5: CRL4 ubiquitylates TOP1-DPCs for proteasomal degradation in a replication-dependent manner.

a HCT116 cells were transfected with empty vector (EV), Myc-CUL4A + RBX1-FLAG overexpression plasmids (CRL4A) or Myc-CUL4B + RBX1-FLAG overexpression plasmids (CRL4B) for 48 h. The cells were subjected to 1 h pre-treatment with replication inhibitor aphidicolin (APH, 10 μM) or CDK7/transcription inhibitor THZ1 (10 μM), followed by co-treatment with CPT (20 μM, 30 min). The cells were subjected to DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. b HCT116 cells were transfected with the Ub K48 or K63 single lysine overexpression plasmid and Myc-CUL4A or B + RBX1-FLAG overexpression plasmids for 48 h before CPT treatment (20 μM, 30 min). The cells were then subjected to DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. c HCT116 transfected with EV or Myc-CUL4A + RBX1-FLAG overexpression plasmids were pre-treated with BTZ (1 μM) or APH (10 μM) for 1 h before co-treatment with CPT (500 nM) for 2 h. The cells were then subjected to ICE assay for immunodetection of TOP1-DPC using anti-TOP1 antibody. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. d Densitometric analyses of TOP1-DPCs from triplicate experiments including blots in panel C. Density of TOP1-DPC/density of DNA of each group was normalized to that of cells transfected with EV only. Data are presented as mean ± SD, N = 3 biologically independent experiments. The p value was calculated using two-tailed Student’s t test. e HCT116 transfected with EV or Myc-CUL4A + RBX1-FLAG overexpression plasmids were pre-treated with BTZ (1 μM) or APH (10 μM) for 1 h before co-treatment with CPT (500 nM) for 2 h. The cells were then subjected to ICE assay for immunodetection of TOP1-DPC using anti-TOP1 antibody. f Densitometric analyses of TOP1-DPCs from triplicate experiments including blots in (e). Density of TOP1-DPC/density of DNA of each group was normalized to that of cells transfected with EV only. Data are presented as mean ± SD, N = 3 biologically independent experiments. The p value was calculated using two-tailed Student’s t test.