Fig 2: RAD9 protein binds the NEIL1 promoter. Binding of RAD9 and Rad9 to their corresponding NEIL1/Neil1 promoter was tested by ChIP-qPCR, using DU145 and mES cells, respectively. (A) Schematic representation of NEIL1/Neil1 promoters with the approximate position of primer pairs used for ChIP-qPCR experiments (see Supplementary Table S1 for primer details); each letter represents the primer pair; asterisk indicates reverse orientation primer of pair. TSS is the transcription start site. (B) Fold enrichment of RAD9 or Rad9 relative to IgG in ChIP-qPCR experiments. Error bars represent the standard deviation of three independent experiments.

Fig 3: Deletion analysis used to determine the region of mouse Rad9 protein involved in Rad9–Neil1 binding. (A) Ectopically expressed RAD9 and endogenous Neil1 were immunoprecipitated from Rad9−/− mES cells individually and tested for binding to the other. (B) Same as A, but in DU145-shRAD9 cells ectopically expressing FLAG-Rad9. (C) Graphic depiction of amino acids encoded by inherent or truncated Rad9 cloned into pCMV6-AC-DDK-His vector, which adds a C-terminal FLAG-His tag to each protein; numbers represent the amino acid positions. Dark box at C-terminal end represents FLAG-His tag. Rad9, full length; Rad9 N, amino-end fragment; Rad9 C, carboxy-end fragment. (D) Binding of intact or deletion mutants of Rad9 to Neil1, shown by IP either with anti-FLAG (upper panel) or anti-Neil1 (lower panel) antibody. (E) Immunoblot showing abundance of Rad9 and Neil1 proteins in whole cell extracts from IP experiments in panel B. β-Actin was used as loading control. Rad9 N, amino-terminal fragment; Rad9 C, carboxy-terminal fragment; NS, non-specific; IP, immunoprecipitation; IB, immunoblot.

Fig 4: Rad9 controls Neil1 protein stability in mES but not in DU145 cells. (A) Neil1 and Rad9 protein levels were detected by immunoblotting in Rad9+/+ and Rad9−/− mES cells after treating with CHX (50 μg/ml) for indicated time intervals. β-Actin was the loading control. (B) Same as A, but using DU145 cells with or without shRAD9. (C) Average Neil1 protein level relative to β-Actin was calculated by densitometric measurements of bands from three independent experiments (as in A, B). Error bars represent standard deviation. (D) Neil1 and Rad9 abundance was assessed by immunoblotting analyses using Rad9+/+ and Rad9−/− mES cells grown in the presence or absence of proteasomal inhibitor MG132 at concentrations indicated. β-Actin is the loading control. (E) Average Neil1 protein level relative to β-Actin was calculated by densitometric measurements of bands from three independent experiments (as in D).

Fig 5: Clonogenic survival of mES and DU145 cells with varying status of Rad9 after UV, menadione, and gamma-ray treatment. (A, B) Sensitivity of cells to 254 nm UV light. (C, D) Sensitivity of cells to menadione. (E, F) Sensitivity of cells to gamma rays. A, C and E, parental DU145 cells or those with shControl or shRAD9, and the latter with insertless pCMV6-AC-DDK-His vector, or ectopically expressing Rad9+ or NEIL1+. B, D and F, mES cells Rad9+/+, Rad9−/−, or the latter with insertless pCMV6-AC-DDK-His vector, or ectopically expressing RAD9+, Rad9+, NEIL1+ or Neil1+. Percent survival after each treatment was calculated as the number of colonies formed in treated versus mock-treated populations, times 100. Points are the average of three independent trials, each with two dishes per point. Error bars, standard deviation.