Nuclear localization of p27Kip1 in response to H₂O₂ scavenging and intrinsic low levels of H₂O₂.

<p>(A-F) Subcellular localization of p27Kip1 evaluated by immunocytofluorescence. (A–B) Melanoma cells treated with 500 and 1000 U/ml catalase (CAT) for periods of 6 or 24 h or left untreated. FBS starved cells were used as control of G1 arrest. (C–D) Catalase overexpression model (A375-CAT-E9 cells) vs. controls (A375-pcDNA3 and A375 control cells). (E-F) Non-tumor (PIG-1) vs. tumor (A375) cells. (A, C and E) Representative images of p27Kip1 immunocytofluorescence showing the subcellular localization of the protein. DAPI: staining of nuclear DNA; p27Kip1: FITC staining of p27Kip1 protein. (B, D and F) Percentage of positive (□) cytoplasms and positive (■) nuclei for p27Kip1 relative to the total number of counted cells. Data are expressed as mean ± SD. (B) **p<0.01 vs. control. (D) *p<0.05 and **p<0.01 vs. A375 control. (F) *p<0.05 and **p<0.01 vs. non-tumor cells. (G–H) Increased expression of nuclear p27Kip1 in A375 cells after 1000 U/ml catalase (CAT) treatment as compared with control A375 cells (treated with 1000 U/ml heat-inactivated catalase, IN-CAT) for 24 h, detected by western blot of nuclear and cytosolic extracts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044502#s4" target="_blank">Methods</a>). (G) Representative immunoblot images are shown. C: Cytoplasmic extracts; N: Nuclear extracts. Actin and Ku-70 densitometric values were used to standardize for cytoplasmic and nuclear protein loading, respectively. (H) Relative densitometric values of (□) cytoplasmic and (■) nuclear p27Kip1 levels. Results are referred to control cells. Data are expressed as mean ± SD. *p<0.05 vs. control.