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. 2016 Aug 5;7(37):60005–60020. doi: 10.18632/oncotarget.11088

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Copyright: © 2016 Du 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 credited.

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(A) Short-lived response of G₁-arrest-associated cell-cycle regulators in response to oncogenic stress in FA HSPCs. LSK cells isolated from Luc-LSL-Fanca^+/+/K-ras/CreER or Luc-LSL-Fanca^−/−/K-ras/CreER mice were cultured in the presence of 4-OHT for 2 hours then released in fresh medium for the indicated time intervals. RNA was extracted at different time points for qPCR analysis using primers listed in Table S1. Levels of the expression in each sample were normalized to the level of GAPDH mRNA, and the expression levels of the Fanca^+/+ samples at 2 h were normalized as 100. (B) p53 is essential for the prolonged oncogenic response in HSPCs. LSK cells isolated from LSL-Fanca^+/+/K-ras/CreER-p53^+/+, LSL-Fanca^−/−/K-ras/CreER-p53^+/+, LSL-Fanca^+/+/K-ras/CreER-p53^−/−, LSL-Fanca^−/−/K-ras/CreER-p53^−/− mice were cultured in the presence of 4-OHT for 2 hours then released in fresh medium for the indicated time intervals. RNA was extracted at different time points for qPCR analysis using primers for p16 (Upper) and p21 (Lower) Samples were normalized to the level of GAPDH mRNA. (C) K-ras activation does not alter p53 level. BM Lin⁻ cells from Luc-LSL-Fanca^+/+/K-ras/CreER or Luc-LSL-Fanca^−/−/K-ras/CreER mice were cultured in the presence of 4-OHT for 2 hours then released in fresh medium for the indicated time intervals. Whole cell lysates were prepared then subjected to immunoblot using antibodies against p53 or β-actin. p53:β-actin ratio in each sample was calculated. The WT control sample at 0 h time point has been normalized as 1. (D) FA deficiency leads to short-lived arginine methylation of p53 in response to oncogene activation. BM Lin⁻ cells from Luc-LSL-Fanca^+/+/K-ras/CreER or Luc-LSL-Fanca^−/−/K-ras/CreER mice were cultured in the presence of 4-OHT for 2 hours then released in fresh medium for the indicated time intervals. Whole cell lysates were prepared, and subjected to immunoprecipitation using anti-p53 antibody followed by immunoblotting with antibodies against phosphor-serine, acetylated lysine, methylated lysine, mono-methyl arginine or p53, respectively.