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. 2011 Aug 24;6(8):e23571. doi: 10.1371/journal.pone.0023571

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Wang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) IRTKS inhibited cell death triggered by serum starvation. The IRTKS Tet-off HT1080 cells were cultured in serum-free medium in the presence or absence of doxycycline (DOX, 10 ng/ml). The cells were collected and analysed by FACS after staining with propidium iodide (PI) at the indicated time. The sub-G1 population was counted as the percentage of cell death. The inducible expression of flag-tagged IRTKS was assessed by Western blotting with anti-FLAG and anti-IRTKS antibodies (right panel). (B, C) IRTKS inhibited UV-induced apoptosis. HT1080 cells with overexpression (B) or knockdown (C) of IRTKS were exposed to UV irradiation and analyzed for apoptosis by Annexin V-FITC/PI (propidium iodide) staining. Representative results of four independent experiments were shown. P<0.005. The expression of IRTKS was assessed by Western blotting (right panel). (D) Overexpression of IRTKS inhibited p53-induced apoptosis. SAOS-2 cells infected with adenoviruses for IRTKS or/and p53 overexpression were analyzed with PI staining by FACS. Data were shown as mean ± s.d. (n = 3), p<0.01. The expression of p53, IRTKS and p53 inducible gene products (PIG3 and MDM2) were analyzed by Western blotting (right panel). (E) Knockdown of IRTKS enhanced p53-induced apoptosis. SAOS-2 cells were treated with IRTKS-siRNA for 24 h, then infected with Ad-p53 or control virus Ad-GFP, and analyzed for apoptosis. Data were shown as mean ± s.d. (n = 3), p<0.01. The expression of p53, IRTKS and p53 inducible gene products (PIG3 and MDM2) were analyzed by Western blotting (right panel). (F) IRTKS inhibited the expression of p53-inducible genes. Total RNA from the IRTKS Tet-off HT1080 cells in the presence (10 ng/ml) or absence of doxycycline was subjected to real-time quantitative PCR (qPCR) analysis. Data were expressed as mean ± s.d. (n = 3). p<0.01. (G) IRTKS decreased the transactivation of p53. p53 transactivation activity on the BAX promoter was measured by luciferase reporter gene assay in SAOS-2 cells.