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. 2013 Feb 14;8(2):e56308. doi: 10.1371/journal.pone.0056308

Figure 7. Regulation of the CDKN1A/p21/WAF mRNA and protein by EVI1, etoposide, and daunorubicin.

Figure 7

A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method [48], with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p<0.05; n.s., not significant (paired Student’s t-test). C) Immunoblot analysis of p21 protein in U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide for 48 h. Hybridization with a β-tubulin antibody was used as a loading control. In the absence of etoposide, p21 is below detection level with the exposure time used. D) Immunoblot analysis of whole cell (WC), cytoplasmatic (cyto), and nuclear (nu) extracts from U937_vec and U937_EVI1 cells. The same amount of protein was loaded for cytoplasmatic and nuclear extracts, corresponding to up to twice as many cell equivalents for the latter. The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls.