Figure 2. Age‐related AR downregulation impairs its stimulatory effect on XRCC4 expression as a transcription factor.

• A
  Putative transcription factors bound to XRCC4 promoter predicted with PROMO algorithm.
• B
  Comparison of the relative AR mRNA level (normalized to GAPDH mRNA level) in cells derived from young and aged individuals. n = 7 biological replicates.
• C
  Analysis of the protein expression level of AR, XRCC4, and Lig4 in the cells derived from young and aged individuals, by Western blot.
• D
  Quantification of the gray scale of AR protein level. Each dot represents the average relative gray scale value of AR (normalized to the gray scale of GAPDH), assayed by three independent repeats of Western blot, for one donor.
• E
  Correlation between the relative AR protein level and donor age. n = 14 donors.
• F
  Correlation between the relative AR protein level and relative XRCC4 mRNA level. n = 14 donors.
• G
  Illustration of the predicted AR‐binding sites on XRCC4 promoter. Arrows indicate the seven pairs of primers designed for amplifying the sequences flanking the eight predicted AR‐binding sites.
• H
  ChIP‐qPCR analysis of relative AR enrichment to the predicted binding sites on XRCC4 promoter. Flag‐tagged AR was transfected into cells, and immunoprecipitation was performed with anti‐Flag or IgG. n = 4 biological replicates.
• I
  ChIP‐qPCR analysis of the enrichment of endogenous AR to XRCC4 promoter. n = 3 biological replicates.
• J
  Analysis of XRCC4 promoter activity in HCA2‐hTERT cells integrated with shRNA against AR, and cells were transfected with empty vector or vectors encoding AR to restoring AR expression. n = 3 biological replicates.
• K
  Schematic diagrams of the firefly luciferase reporter driven by the wild‐type XRCC4 promoter and the promoter with AR‐binding site TGCATGTCC (−166 bp to −158 bp) deleted.
• L
  The effect of AR overexpression on XRCC4 promoter activity, assayed with the two luciferase reporters in (K). n = 3 biological replicates.
• M
  The effect of AR overexpression on the protein level of XRCC4 and Lig4. EV, empty vector.
• N
  The effect of AR knockdown on the protein level of XRCC4 and Lig4.
• O, P
  The effect of shRNA‐mediated knockdown of AR on the genomic stability in HCA2‐hTERT cells, analyzed by comet assay. Each dot represents one comet analyzed (n = at least 150 cells). Representative comet images are shown in (O), and the quantitative result is shown in (P).

Error bars represent the s.e.m. values in (B), (D) to (F), (H) to (J), and (L). Mann–Whitney U test was performed to determine the significance of the results presented in (B), (D), and (P), unpaired t test was performed to determine the significance of the results presented in (H), (I), (J), and (L), and t test was performed to assess the significance of the regression slope in (E) and (F).

Source data are available online for this figure.