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. 2012 Oct 2;7(10):e46567. doi: 10.1371/journal.pone.0046567

Figure 2. Erioflorin stabilizes Pdcd4 without affecting phosphorylation events.

Figure 2

(A + B) HEK293 cells were transiently transfected with Pdcd4(39–91)luc (A) or Pdcd4(mut39–91)luc (B) firefly reporter vectors, in combination with expression vectors for either wildtype (S6Kwt = white bars) or constitutively active p70S6K (S6Kca = black bars) and a renilla luciferase vector one day prior to the experiment. Transfected cells were treated for 8 h with rapamycin (100 nM) or erioflorin (5 µM). Firefly normalized to renilla luciferase activity is presented relative to DMSO-treated controls. (C) HEK293 cells were treated for 8 h with TPA (10 nM) with or without erioflorin (0.625–5 µM). Whole-cell extracts were subjected to western analysis and probed with the indicated antibodies. Blots are representative of at least three independent experiments. Densitometric analysis and quantification of nucleolin-normalized Pdcd4 and phospho-S6 protein levels is shown relative to the DMSO control. (D) HEK293 cells were treated with DMSO (black diamonds), TPA (10 nM) with (white triangles) or without (gray squares) erioflorin (5 µM) for 8 h and cycloheximide (10 µM) was added for 1, 2 or 4 h. Pdcd4 protein levels were analyzed densitometrically, normalized to nucleolin and the half-life was calculated. All data are presented as means ± SEM (n≥3, *p<0.05, **p<0.01, ***p<0.001).