Rad51 forms foci following treatment with 0.4 μM aphidicolin. (A) HeLa cells were treated with 0.4 μM aphidicolin for 24 h, fixed, and stained with anti-Rad51 antibodies (α-Rad51). Untreated cells were analyzed as control. (B) Number of Rad51 nuclear foci in cells treated with 0.4 μM aphidicolin and in untreated cells. The data presented are based on at least two independent samples.

Rad51 down-regulation by RNAi leads to increased common fragile site expression. (A) Western blot probed with α-RAD51 in MCF7 cells transfected with pSUPER encoding siRNA directed against Rad51 (pS-Rad51). Transfection with the pSUPER plasmid (pS) was analyzed as control. Reduction in protein level was 90%. (B) Number of gaps and constrictions per metaphase in MCF7 cells transfected with pS-Rad51 with or without treatment with 0.4 μM aphidicolin for 24 h. Transfection with pS was analyzed as control. (C) Example of a metaphase from MCF7 cells transfected with pS-Rad51 showing a high number of gaps and constrictions (n = 15). The box in the bottom right is a magnification of the area marked in the picture. Arrows mark gaps and constrictions. (D) Frequency of fragile site (FS) FRA3B and FRA16D expression with or without 0.4 μM aphidicolin treatment for 24 h in MCF7 cells transfected with pS-Rad51or pS. Error bars indicate the standard error. The data presented are based on at least two independent samples.

Ligase IV down-regulation by RNAi leads to increased common fragile site expression. (A) Western blot probed with α-Ligase IV in MCF7 cells transfected with pSUPER encoding siRNA directed against Ligase IV (pS-LigIV). Transfection with the pSUPER plasmid (pS) was analyzed as control. Reduction in protein level was 90%. (B) Number of gaps and constrictions per metaphase in MCF7 cells transfected with pS-LigIV with or without treatment with 0.4 μM aphidicolin for 24 h. Transfection with pS was analyzed as control. (C) Example of a metaphase from MCF7 cells transfected with pS-LigIV showing a high number of gaps and constrictions (n = 18). The box in the top left is a magnification of the area marked in the picture. Arrows mark gaps and constrictions. (D) Frequency of fragile site (FS) FRA3B and FRA16D expression following treatment with 0.4 μM aphidicolin for 24 h in MCF7 cells transfected with pS-LigIV or pS. Error bars indicate the standard error. The data presented are based on at least two independent samples.

MDC1 forms foci following treatment with 0.4 μM aphidicolin. (A) HeLa cells were treated with 0.4 μM aphidicolin for 24 h, fixed, and stained with anti-MDC1 antibodies (α-MDC1). Untreated cells were analyzed as control. (B) Number of MDC1 nuclear foci in cells treated with 0.4 μM aphidicolin and in untreated cells. The data presented are based on at least two independent samples.

DNA-PKcs forms foci following aphidicolin treatment. (A) HeLa cells were treated with the indicated concentration of aphidicolin for 24 h, fixed, and stained with antibodies against the DNA-PKcs, phosphorylated on Thr2609 (α-pDNA-PKcs). Untreated cells were analyzed as control. (B) Number of phospho-DNA-PKcs nuclear foci in cells treated with 0.4 μM aphidicolin and in untreated cells. (C) Number of phospho-DNA-PKcs nuclear foci in cells treated with the indicated aphidicolin concentration. Note that the categories are different from those in B to allow comparison with the high number of foci obtained with 6 μM aphidicolin. (D) Number of gaps and constrictions per metaphase in HeLa cells treated for 24 h with the indicated aphidicolin concentration. (E) HeLa cells were treated with 0.4 μM aphidicolin for 24 h, fixed, and costained with α-pDNA-PKcs and α-Rad51. The data presented are based on at least two independent samples.

DNA-PKcs down-regulation by RNAi leads to increased common fragile site expression. (A) Western blot probed with α-DNA-PKcs in MCF7 and HeLa cells transfected with pSUPER encoding siRNA against DNA-PKcs (pS-D-PK). Transfection with the pSUPER plasmid (pS) was analyzed as control. Reduction in protein level was 85% and 65% for MCF7 and HeLa cells, respectively. (B) Number of gaps and constrictions per metaphase in MCF7 cells transfected with pS-D-PK with or without treatment with 0.4 μM aphidicolin for 24 h. Transfection with the pS plasmid was analyzed as control. (C) Number of gaps and constrictions per metaphase in HeLa cells transfected with pS-D-PK with or without treatment with 0.4 μM aphidicolin for 24 h. Transfection with the pS was analyzed as control. (D) Example of a metaphase from MCF7 cells transfected with pS-D-PK showing a high number of gaps and constrictions (n = 28). The box in the top right is a magnification of the area marked in the picture. Arrows mark gaps or constrictions. (E) Frequency of fragile site (FS) FRA3B and FRA16D expression following treatment with 0.4 μM aphidicolin for 24 h in MCF7 cells transfected with pS-D-PK. Transfection with the pS was used as control. (F) The same experiment as in E, performed in HeLa cells. Error bars indicate the standard error. The data presented are based on at least two independent samples.

γH2AX forms foci following aphidicolin treatment. (A) HeLa cells were treated with the indicated aphidicolin concentrations for 24 h, fixed, and stained with anti-γH2AX antibodies (α-γH2AX). Untreated cells were analyzed as control. (B) Number of γH2AX nuclear foci in cells treated with 0.4 μM aphidicolin and in untreated cells. (C) Number of γH2AX nuclear foci in cells treated with the indicated aphidicolin concentration. Note that the categories are different from those in B to allow comparison with the high number of foci obtained with 6 μM aphidicolin. (D) HeLa cells were treated with 0.4 μM aphidicolin for 24 h, fixed, and costained with α-γH2AX and α-pDNA-PKcs. (E) HeLa cells were treated with 0.4 μM aphidicolin for 24 h, fixed, and costained with α-γH2AX and α-Rad51. The data presented are based on at least two independent samples.

γH2AX and phospho-DNA-PKcs foci localize to expressed fragile sites at metaphase. Chromosomes from HeLa cells treated with 0.4 μM aphidicolin for 24 h were stained with anti-γH2AX or anti-phospho-DNA-PKcs and hybridized with a probe from the FRA3B region. (A) DAPI staining of chromosome 3 expressing FRA3B (arrow). (B) The same chromosome showing a γH2AX immunofluorescent signal (red) and a FISH signal of a probe from the FRA3B region (green). (C) DAPI staining of chromosome 3 expressing FRA3B (arrow). (D) The same chromosome showing a phospho-DNA-PKcs immunofluorescent signal (red) and a FISH signal of a probe from the FRA3B region (green). (E) Quantitation of the localization of γH2AX and phospho-DNA-PKcs foci to the FRA3B region. Error bars indicate the standard error. The data presented are based on at least two independent samples.