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. 2006 Feb;26(4):1398–1413. doi: 10.1128/MCB.26.4.1398-1413.2006

FIG. 4.

FIG. 4.

Knockdown of PolH impairs p53 activation and activity upon DNA damage. (A) p53 activation is impaired in RKO-PolH-KD cells. RKO and its derivatives were treated with 100 nM CPT for 6 h. Cell extracts were collected, and p53, PolH, and actin were detected by Western blot analysis. (B) p53 activation is decreased by inducible PolH knockdown in RKO cells treated with CPT. RKO cells that were uninduced or induced to knock down PolH were treated with CPT at indicated concentrations for 6 h. Cells were collected, and p53, PolH, and actin were detected by Western blot analysis. (C) p53 activation is decreased by inducible PolH knockdown in RKO cells following exposure to IR. RKO cells that were uninduced or induced to knock down PolH were irradiated with 2 Gy of IR. At 4 h after IR, cells were collected, and p53, PolH, and actin were detected by Western blot analysis. (D) Impaired p53 activation by PolH knockdown is not cell type specific. MCF-7 and MCF-7-PolH-KD#43 cells were treated with 100 nM CPT for 6 h. Cell extracts were collected, and p53, PolH, and actin were detected by Western blot analysis. (E) p53 activation is decreased in PolH-null human fibroblasts following exposure to IR. Normal human fibroblasts (GM00495 and GM00024) and human XPV fibroblasts (GM03617 and GM02359) were irradiated with 2 Gy of IR. At 10 h following IR, cells were collected and p53, PolH, and actin were detected by Western blot analysis. (F) The induction of p21 and Fas by DNA damage is decreased in PolH knockdown cells. RKO and its derivatives were treated with 300 nM CPT for 6 h. The expression of p53, PolH, p21, Fas, and actin was examined by Western blot analysis. (G) The induction of DR5 and Mdm2 by DNA damage is decreased when PolH is knocked down. RKO cells that were uninduced or induced to knock down PolH were treated with 50 nM CPT for 6 h. Cells were collected, and DR5, Mdm2, and actin were detected by Western blot analysis. (H) The induction of DDB2 by DNA damage is decreased in PolH knockdown cells. RKO and its derivatives were treated with 100 nM CPT for 0, 6, and 9 h. The expression of DDB2 and GAPDH was examined by Northern blot analysis.