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. 2014 Aug 7;42(15):9908–9924. doi: 10.1093/nar/gku705

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 7. — Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. (a) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. (b) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. (c) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. (d) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. (e) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.