Eric aberrations (Figure 4B). Taken together, these results indicated that the vast majority of APH-induced chromatid breaks in immortalized cells without HPV16 E6E7 expression were repaired by end-joining, so that few further chromosomal rearrangements or deletions were detected 72 h after APH removal. The results also excluded the possibility that the preferential pericentromeric instability in HPV16 E6E7-hTERTexpressing cells was mainly due to hTERT expression.Centromeric Instability after Replication StressFigure 3. Chromosomal aberrations 72 h after release from APH treatment. A: Frequencies of non-clonal chromosomal aberrations. B: Examples of pericentromeric chromosomal aberrations. Centromeric regions were identified by the centromeric constrictions, intenseDAPI staining and pan-centromere FISH (green). First panel: An example of pericentromeric chromosomal deletion. Second panel: An example of pericentromeric chromosomal breaks with both arms present. Third panel: An example of pericentromeric chromosomal translocation. Note that the joined region was at centromeric constriction region with centromere FISH signals. Lowest panel: An example of dicentrics with joined regions involving centromeric ends (Xp and 21p). doi:10.1371/journal.pone.0048576.gCentromere-adjacent Large c-H2AX Foci were more Frequently Detected in HPV16 E6E7-hTERT-immortalized than hTERT-immortalized Cells Before and After APH Treatmentc-H2AX is a commonly used DNA damage/response marker. We performed dual-color immunofluorescence staining with antibodies MedChemExpress JI-101 against c-H2AX and centromeric proteins to examine whether the DNA damage/response signals were localized at or near centromeres. Analysis with confocal microscopy showed that significantly greater numbers of large nuclear c-H2AX foci (at least twice as large as centromeric protein foci) were present in HPV16 E6E7-hTERT-immortalized cells than in hTERT-immortalized cells of the same cell origins (P,0.05) (Figure 5). The majority (,70 ) of the large c-H2AX foci were juxtaposed or colocalized with centromeres, as exemplified in Figure 6. At the end of 24 h APH treatment, increased numbers of large c-H2AX foci, together with numerous small c-H2AX foci, were observed in HPV16 E6E7-hTERT-immortalized cells as well as in hTERT-immortalized cells (Figure 6). Seventy-two hours after removal of APH, mainly large c-H2AX foci remained, most ofwhich (,80 ) were juxtaposed with centromeres (Figure 6); and there were significantly more such foci in HPV16 E6E7-hTERTimmortalized cells than in hTERT-immortalized cells (P,0.05, Figure 5).HPV16 E6E7-hTERT-expressing Cells were Deficient in Recovering from Replication Stress-induced S-phase Arrest Compared with hTERT-expressing Triptorelin custom synthesis CounterpartsCell cycle distributions were analyzed using flow-cytometrical analyses (Figure S4). HPV16 E6E7-hTERT-immortalized and hTERT-immortalized cells did not differ remarkably in the partial S-phase arrest (percentages of S-phase increase) at the end of APH treatment. Yet, 72 h after removal of APH, the proportions of Sphases in hTERT-immortalized cells returned almost to the original levels before treatment, whereas those in HPV16 E6E7hTERT-immortalized cells were restored to only half of the original levels. This indicated that HPV16 E6E7-hTERT-expressing cells had slower S-phase recovery rates than hTERTimmortalized cells after release from replication stress.Centromeric Instability after Replication StressFigure 4. Chromosome aberrations after.Eric aberrations (Figure 4B). Taken together, these results indicated that the vast majority of APH-induced chromatid breaks in immortalized cells without HPV16 E6E7 expression were repaired by end-joining, so that few further chromosomal rearrangements or deletions were detected 72 h after APH removal. The results also excluded the possibility that the preferential pericentromeric instability in HPV16 E6E7-hTERTexpressing cells was mainly due to hTERT expression.Centromeric Instability after Replication StressFigure 3. Chromosomal aberrations 72 h after release from APH treatment. A: Frequencies of non-clonal chromosomal aberrations. B: Examples of pericentromeric chromosomal aberrations. Centromeric regions were identified by the centromeric constrictions, intenseDAPI staining and pan-centromere FISH (green). First panel: An example of pericentromeric chromosomal deletion. Second panel: An example of pericentromeric chromosomal breaks with both arms present. Third panel: An example of pericentromeric chromosomal translocation. Note that the joined region was at centromeric constriction region with centromere FISH signals. Lowest panel: An example of dicentrics with joined regions involving centromeric ends (Xp and 21p). doi:10.1371/journal.pone.0048576.gCentromere-adjacent Large c-H2AX Foci were more Frequently Detected in HPV16 E6E7-hTERT-immortalized than hTERT-immortalized Cells Before and After APH Treatmentc-H2AX is a commonly used DNA damage/response marker. We performed dual-color immunofluorescence staining with antibodies against c-H2AX and centromeric proteins to examine whether the DNA damage/response signals were localized at or near centromeres. Analysis with confocal microscopy showed that significantly greater numbers of large nuclear c-H2AX foci (at least twice as large as centromeric protein foci) were present in HPV16 E6E7-hTERT-immortalized cells than in hTERT-immortalized cells of the same cell origins (P,0.05) (Figure 5). The majority (,70 ) of the large c-H2AX foci were juxtaposed or colocalized with centromeres, as exemplified in Figure 6. At the end of 24 h APH treatment, increased numbers of large c-H2AX foci, together with numerous small c-H2AX foci, were observed in HPV16 E6E7-hTERT-immortalized cells as well as in hTERT-immortalized cells (Figure 6). Seventy-two hours after removal of APH, mainly large c-H2AX foci remained, most ofwhich (,80 ) were juxtaposed with centromeres (Figure 6); and there were significantly more such foci in HPV16 E6E7-hTERTimmortalized cells than in hTERT-immortalized cells (P,0.05, Figure 5).HPV16 E6E7-hTERT-expressing Cells were Deficient in Recovering from Replication Stress-induced S-phase Arrest Compared with hTERT-expressing CounterpartsCell cycle distributions were analyzed using flow-cytometrical analyses (Figure S4). HPV16 E6E7-hTERT-immortalized and hTERT-immortalized cells did not differ remarkably in the partial S-phase arrest (percentages of S-phase increase) at the end of APH treatment. Yet, 72 h after removal of APH, the proportions of Sphases in hTERT-immortalized cells returned almost to the original levels before treatment, whereas those in HPV16 E6E7hTERT-immortalized cells were restored to only half of the original levels. This indicated that HPV16 E6E7-hTERT-expressing cells had slower S-phase recovery rates than hTERTimmortalized cells after release from replication stress.Centromeric Instability after Replication StressFigure 4. Chromosome aberrations after.