Fig 2: The POLI treatment enhanced the radioresistance of esophageal squamous cell carcinoma cells in vitro and in vivo.A ESCC patients were classified into low- and high-POLI expression groups according to IHC scores (IHC score 6 was used as a cutoff value for POLI) in all cases. Scale bar, 50 μm. P value was measured by Student’s t test. B Results recorded from Kaplan-Meier survival analysis for overall survival (OS) and disease-free survival (DFS) revealed significantly shortened OS (P = 0.029) and DFS (P = 0.014) for patients with high POLI expression compared to patients with low POLI expression. C The protein levels of POLI in ESCC and normal cell lines were detected by western blot. D Analysis of POLI expression in the ectopic POLI (TE-1-POLI oe vs TE-1-empty) and specific endogenous POLI knockdown (KYSE-150-shPOLI vs KYSE-150-shNC) ESCC cells using Western blot. E Clonogenic survival curves were generated for TE-1 and KYSE-150 cells that were exposed to 2, 4, 6, and 8 Gy X-ray irradiation. The single hit multiple target radiobiological model was used to fit the survival curves. F Images as well as growth curve of the volume out of ESCC tumor xenografts in nude mice. The tumor volumes were presented as the mean ± SEM of three independent experiments. P value was measured by Student’s t test. G The expression level of Ki67 in tumors from the mice was detected by IHC staining. ***P < 0.00, **P < 0.01.

Fig 3: POLI inhibits ubiquitin-mediated degradation of RAD51 by blocking the interaction between RAD51 and XIAP in a competitive binding manner.A Over-expression of POLI impaired the interaction between XIAP and RAD51 proteins. B Schematic diagram of the POLI protein domains. Characterize the regions of POLI mediating its binding with XIAP. C Schematic diagram of RAD51 protein domains. D Characterize the regions of RAD51 mediating its binding with XIAP. E Schematic diagram of XIAP protein domains. F Identification of the regions of XIAP that mediate its binding with POLI and RAD51.

Fig 4: POLI interacts with XIAP and prevents the ubiquitination and degradation of RAD51.A The results of MS analysis suggested that POLI could interact with E3 ligase XIAP. B The immunoprecipitation assay was used to verify the interaction between XIAP and POLI or RAD51 using whole cell lysate from TE-1 cells. C Western blotting was analysis of RAD51 and XIAP expression in the specific endogenous XIAP knockdown ESCC cells. D The specific endogenous XIAP knockdown TE-1 cells and the POLI TE-1 cells with ectopic expression were treated with cycloheximide (CHX, 50 μg/ml) for the indicated times and western blotting was performed to detect RAD51 protein levels. E Western blotting was performed to detect RAD51 protein levels in down-regulating XIAP of TE-1-POLI oe and TE-1-empty cells treated with cycloheximide (CHX, 50 μg/ml). F Western blotting following immunoprecipitation assay was performed to detect the signal of polyubiquitinated RAD51 in the specific endogenous XIAP knockdown TE-1 cells and the POLI TE-1 cells with ectopic expression that were exposed to MG132 (30 μM) for 6 h. G Western blotting was performed after immunoprecipitation to detect polyubiquitinated RAD51 protein levels in down-regulating XIAP of TE-1-empty cells and TE-1-POLI-oe cells.

Fig 5: The loss of POLI enhances the IR-induced immune response in ESCC cells.A, B Quantitative RT-PCR was performed to detect IL1A and IL-32 in ESCC cells expressing either down-regulated or over-expressed POLI. (C) Western blotting was used to detect the expression of p-STAT3, and p-STING in eESCC cells at 72 h following 10 Gy IR treatment. D, E Immunofluorescent staining of cGAS micronuclei was adopted at 72 h after 10 Gy IR. We measured the number of cGAS-positive micronuclei per cell. Scale bar, 100μm. The data was represented as mean ± SEM. P value measured by paired Student’s t test; **P < 0.01.