1. Volume 120, Issue 6, Pages 747-759 (March 2005)
The mTOR Inhibitor RAD001 Sensitizes Tumor Cells to DNA-Damaged Induced Apoptosis through Inhibition of p21 Translation 
Iwan Beuvink, Anne Boulay, Stefano Fumagalli, Frederic Zilbermann, Stephan Ruetz, Terence O’Reilly, Francois Natt, Jonathan Hall, Heidi A. Lane, George Thomas 
Cell 
Volume 120, Issue 6, Pages (March 2005)
DOI: /j.cell
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2. Figure 1 RAD001-Enhanced Cisplatin-Induced Apoptosis Is p53 Dependent
(A and B) A549 cells were treated for 24 hr with either DMSO or 20 nM RAD001 in combination with cisplatin. Proliferation rates and loss of cell viability were measured using the YO-PRO assay (Experimental Procedures). Data represent the mean ± standard deviation of three independent experiments (* = significant fold induction with p < 0.05; t tests and two-way ANOVA indicate that the interaction between RAD001 and cisplatin was highly significant [p < 0.001]).
(C) A549 cells were treated for 24 hr with either DMSO or 20 nM RAD001 in combination with the indicated concentrations of cisplatin. The expression levels of p53 and PARP were assessed by immunoblotting with indicated antibodies.
(D) A549 cells were untransfected or transfected with LacZ or p53 siRNAs (Experimental Procedures) and treated with the indicated doses of cisplatin. Protein levels of p53, PARP, p21, and α-tubulin were assessed by Western blotting.
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3. Figure 2 Rapamycin-Resistant mTOR Protects A549 Cells from RAD001
(A) A549 cells infected with the empty retrovirus (pBabe Puro) or (B) a retrovirus encoding either hemagglutinin-tagged wild-type mTOR (wt) or (C) HA-tagged rapamycin resistant mTOR (RR).
(A) A549 parental, pBabe Puro, (B) HA-mTOR wt clone 15 and 28, as well as (C) the RR clones 11 and 52 were exposed for 24 hr to DMSO, 0.2 nM or 20 nM RAD001. Cell lysates were analyzed by Western blot analysis.
Cell  , DOI: ( /j.cell )
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4. Figure 3
RAD001 Mediates Enhanced Sensitivity to Cisplatin through mTOR
(A) A549 parental, pBabe Puro, HA-mTOR wt (clone 15/28), and HA-mTOR RR (clone 11/52) cells were treated with either DMSO (black bars), 0.2 nM RAD001 (white bars), or 20 nM RAD001 (gray bars). After 4 days, cells were counted, and the data represent the mean ± standard deviation of three independent experiments (* = significant inhibition with p < 0.05, one-way ANOVA).
(B) A549 HA-mTOR wt clones 15 (open circle)/28 (open diamond) and RR clones 11 (filled triangle)/52 (filled square) were treated with the indicated concentration of RAD001. After 3 days, cell proliferation rates were assessed using the YO-PRO assay (Experimental Procedures) and plotted as % of DMSO control-treated cells. Data represent the mean ± standard deviation of three independent experiments.
(C) A549 HA-mTOR wt28 cells and (D) A549 HA-mTOR RR52 cells were treated with the indicated concentrations of cisplatin in the presence of DMSO (black bars) or 20 nM RAD001 (white bars). After 24 hr, cell viability was assessed using the YO-PRO-method. Data represent the mean ± standard deviation of three independent experiments (* indicates statistically significant inhibition at p < 0.05, t tests and two-way ANOVA indicates that the interaction between RAD001 and cisplatin was highly significant [p < 0.001]).
Cell  , DOI: ( /j.cell )
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5. Figure 4
RAD001 Inhibits Cisplatin-Induced p21 Expression in A549 and MCF7 Cells
(A) A549 and (B) MCF7 cells were treated for 24 and 30 hr, respectively, with indicated concentrations of cisplatin in the presence of DMSO or 20 nM RAD001. Protein levels of p21, Bax, and actin were assessed by Western blotting.
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6. Figure 5
RR mTOR Protects Cisplatin-Induced p21 Downregulation by RAD001
(A) A549 HA-mTOR wt28 cells or (B) A549 HA-mTOR RR52 cells were treated for 24 hr with indicated concentrations of cisplatin in the presence of DMSO or 20 nM RAD001. PARP, p21, and actin protein levels were assessed by Western blotting.
Cell  , DOI: ( /j.cell )
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7. Figure 6 Activation of p53-Dependent Anti- and Proapoptotic Programs
(A) A549 cells treated with 1 or 15 µg/ml cisplatin were incubated for the indicated times. PARP, p53, p21, and actin protein levels were assessed by Western blotting.
(B) A549 cells were untransfected or transfected with 100 nM LacZ or p21siRNAs. After 30 hr, cells were treated for 24 hr with the indicated concentrations of cisplatin. PARP, p53, p21, and actin protein levels were assessed by Western blotting.
Cell  , DOI: ( /j.cell )
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8. Figure 7
RAD001 Blocks p21 Expression by Inhibition of Global Translation
(A) A549 cells were treated for 24 hr with 0.5 µg/ml cisplatin in the presence of DMSO or 20 nM RAD001, followed by cycloheximide for the indicated times. Left panel, a representative experiment, in which actin and p21 protein levels were assessed by Western blotting. Right panel, the fractional signal loss of p21 protein was determined using ImageQuant. The data represents the mean ± standard deviation of five independent experiments. p21 half-life (t1/2) was calculated for each individual sample (n = 5) using nonlinear regression (one phase exponential decay). The differences between the t1/2 of DMSO- and RAD001-treated cells, 0.7 ± 0.1 hr and 0.5 ± 0.07 hr, respectively, was not significant (p = 0.336; t test).
(B) After 24 hr, total RNA was isolated and p21, β-actin, and 18S RNA levels were assessed by Northern blot analysis with the indicated probes from A549 cells treated with or without 0.5 µg/ml cisplatin in the presence of DMSO or 20 nM RAD001.
(C) A549 cells were treated for 24 hr with either 0.5 µg/ml cisplatin alone or in combination with 20 nM RAD001. Cell extracts were fractionated on sucrose gradients and mRNAs encoding eEF-1α (top left panel), β-actin (bottom left panel), and p21 (right panel) located by Northern blot analysis with the indicated probe.
(D) Northern blot and analysis of p21 mRNA located on polysome profiles displaying a more shallow gradient. ([C], right panel, and [D]) Gradient analysis of the polysome profiles of A549 cells treated 24 hr with 0.5 µg/ml cisplatin alone (black lines) or in combination with 20 nM RAD001 (gray lines).
(C and D) polysomes and (D) 40S subunits, 60S subunits, and 80S ribosomes are indicated.
Cell  , DOI: ( /j.cell )
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