p21 regulates G₁/S arrest and cell survival under conditions of amino acid deprivation.

<p>A) Western blot analysis of the MEFs used in (B) to determine p21 knockdown efficiency. Values below blot represent the fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. β-tubulin was used as a loading control. B) G_<b>1</b>/S ratio of leucine-starved GCN2^+/+ MEFs stably transfected with non-targeting shRNA (shNT) or shRNA against p21 (shp21). DNA content was measured by propidium iodide staining and flow cytometry analysis. Data are the average of three independent experiments ± S.E.M.; * p<0.05, ** p<0.01. C) Western blot analysis of the SQ20Bs used in (D) to determine p21 knockdown efficiency. Values below blot represent the fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. β-tubulin was used as a loading control. D) G_<b>1</b>/S ratio of leucine-starved SQ20Bs transfected with non-targeting siRNA (siNT) or siRNA against p21 (sip21). DNA content was measured by propidium iodide staining and flow cytometry analysis. Data are the average of four (siNT) or five (sip21) independent experiments ± S.E.M.; * p<0.05, ** p<0.01. E) Clonogenic survival of control and p21 knockdown GCN2^+/+ MEFs exposed to long-term leucine starvation. Data are the average of three independent experiments ± S.E.M; *p< 0.5, **p< 0.1. F) Representative picture of control and p21 knockdown MEFs from the clonogenic survival assay described in (E) after one week of colony formation.