Fig 2: Overexpression of RAD18 increases the metastatic potential of CRC cells via the EMT pathway. (A) EMT biomarkers, including E-cadherin, N-cadherin, vimentin and RAD18 were detected by western blot analysis in HCT116 LV3 cells, HCT116 RAD18sh (RAD18 knockdown), DLD-1 LV3, DLD-1 RAD18sh (RAD18 knockdown), SW480 LV5 and SW480 RAD18 (RAD18-overexpressing) cell lines. All the experiments were repeated three to four times with similar findings. Band intensity was quantified by ImageJ software and are shown by a histogram. *P<0.01, ***P<0.001, Student's t-test. (B) Immunohistochemical analysis of RAD18, E-cadherin, N-cadherin and vimentin in CRC tissues. Representative patients, RAD18 (+) and RAD18 (−) were selected from 93 patients with CRC (Scale bar, 100 µm; magnification, ×200). (C) E-cadherin, N-cadherin, vimentin and RAD18 expression was detected in 93 CRC tissues by PCR. RAD18 expression was positively correlated with N-cadherin and vimentin expression (P<0.001), but negatively correlated with E-cadherin expression (P<0.001). EMT, epithelial-mesenchymal transition; RAD18, E3 ubiquitin protein ligase; CRC, colorectal cancer.

Fig 3: Hypothetical model. DDUP encoded by an lncRNA orchestrates DNA damage repair by regulating PCNA monoubiquitination and RAD18 retention at the DNA damage sites.

Fig 4: Cezanne localizes to DNA damage sites and promotes Rad18 IRIF. (A) GFP-Cezanne, but not the Cezanne mutant lacking the UBA domain, accumulates to laser-induced DNA damage. U2OS cells stably expressing GFP-Cezanne WT or ΔUBA treated with laser microirradiation were fixed and stained. (B) Cezanne deficiency leads to decreased Rad18 IRIF. U2OS or U2OS stably expressing GFP-RNF168 cells were treated with control or Cezanne siRNA, irradiated with 10 Gy IR, followed by 2 h incubation before fixation and staining. Student's t-test was used for statistical analysis.

Fig 5: DDUP enhances the retention of RAD18 at DNA damage sites. (A) Representative images (left) and time course (right) of the formation of CPT (10 μM)-induced RAD18 and RAD51C foci in control and DDUP-KO HeLa cells and allowed to recover for the indicated times. The RAD18- and RAD51C foci was examined every 10 min in the CPT-treated cells within the first 2 h. Cells containing more than 10 RAD18 and RAD51C foci per nucleus were scored. (B) Chromatin fraction and IB analysis of DNA-bound RAD18, RAD51C and DDUP in the indicated CPT (10 μM)-treated cells and allowed to recover for the indicated times. H3 served as a loading control. (C) Quantitative FRAP analysis of GFP-RAD18 in GFP-RAD18-transfected control and DDUP-KO HeLa cells (right), and in DDUP-KO HeLa cells co-transfected with GFP-RAD18 and vector, GFP-RAD18, and DDUP/WT, or GFP-RAD18 and DDUP/T174A, treated with CPT (10 μM) and allowed to recover for the indicated times. (D) Kinetics of γ-H2AX signals in the indicated cells in response to laser micro-irradiation and allowed to recover for the indicated times (n = 100). (E) IP assay analysis of the DDUP/RAD51C and DDUP/PCNA interaction in control and RAD18-silenced 293T cells treated with CDDP (5 μM, 1 h). (F) Homologous recombination repair assays performed in the indicated cells. (G) IP/IB analysis of the regulatory effect of DDUP dysregulation on PCNA monoubiquitination in the indicated cells treated with CDDP (5 μM, 1 h) or UV radiation (60 J/m2). H3 and α-tubulin served as loading control. Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).