Fig. 3: Neddylation fosters the repair of TOP1-DPCs in a manner epistatic to the ubiquitin-proteasome pathway. | Nature Communications

Fig. 3: Neddylation fosters the repair of TOP1-DPCs in a manner epistatic to the ubiquitin-proteasome pathway.

From: Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex

Fig. 3

a Scheme of the in vivo complex of enzyme (ICE) bioassay. b Left panel: ICE bioassay shows that SN38-induced TOP1 were removed after 4 h exposure to SN38 in HCT116 cells, and that pre-treatment with 10 µM PEV for 1 h blocked the removal of TOP1-DPCs. DNA loading of each sample was confirmed using anti-DNA antibody. Right panel: densitometric analyses of TOP1-DPCs from triplicate experiments including blots in the left panel. Density of TOP1-DPC/density of DNA of each group was normalized to that of cells treated with 500 nM SN38 for 1 h. Data are presented as mean ± SD, N = 3 biologically independent experiments. The p values in this figure were calculated using two-tailed Student’s t test. c Top panels: 20 s filming of TOP1-HaloTag single molecules in HCT116 cells. The cells were divided into indicated treatments. Middle panels: plots of tracks of TOP1-HaloTag single molecules as shown in the top panels. Bottom panels: count of jumps of TOP1-HaloTag single molecules derived from the top-panel films. The bin size is 0.1 μm. d Left panel: ICE bioassay in HCT116 cells treated with the indicated drug combinations for 2 h. Right panel: densitometric analyses of TOP1-DPCs. Density of TOP1-DPC/density of DNA of each group was normalized to that of cells treated with SN38 alone. Data are presented as mean ± SD, N = 3 biologically independent experiments. e HCT116 cells were treated with SN38 (10 μM) and collected at indicated time points for Western blotting. Cells were then lysed with the neutral lysis procedure. The scale bar represents 300 μm. f Left panel: HCT116 were treated with SN38 (10 μM) for 2 h in the presence and absence of indicated inhibitors. Cells were then subjected to neutral comet assay. Right panel: quantitation of tail moments for comet assay samples using OpenComet. Data are presented as mean ± SD, n = 180 total cells. Biological independent experiments were repeated three times. g HCT116 cells were treated with SN38 (10 μM) for 2 h. PEV (10 μM) and BTZ (1 μM) were added 1 h prior to the SN38 treatment. Cells were then subjected to Western blotting using indicated antibodies.

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