Cyp24a1 contains an IRES element.

<p>(A) Sequence of the human cyp24a1-5′UTR. (B) Schematic representation of the bicistronic control (phpRF) and cyp24a1-5′UTR-containing (phpR-cyp-F) luciferase constructs used for reporter assays. (C) Bicistronic reporter plasmids phpRF (white bars) and phpR-cyp-F (black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured and data are presented as means ± SEM relative to phpRF (n≥3, ** p<0.01). (D) RNA isolated from cells transfected with phpRF or phpR-cyp-F was DNAse treated and reverse transcribed. <i>Upper panel</i>: PCR was performed with specific primers to amplify full length RL or R-cyp-L mRNAs. PCR products were visualized <i>via</i> agarose gel electrophoresis and ethidium bromide staining. Data are representative for at least 3 independent experiments. <i>Lower panel</i>: RT-qPCR analysis of the amount of <i>firefly</i> mRNA normalized to <i>renilla</i> mRNA. Data are presented as means ± SEM (n≥3). (E) <i>In vitro-</i>transcribed mRNAs of the control (hpRF, white bars) or the cyp24a1-5′UTR-containing vector (hpR-cyp-F, black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured. Luciferase activities are given relative to hpRF and data are presented as means ± SEM (n≥3, ** p<0.01).</p

Polysome profile of MCF7 cells.

<p>Representative profile of MCF7 lysates at 254 nm as determined during polysomal fractionation (<i>upper panel</i>). Equal aliquots of RNA isolated from single fractions were analyzed using denaturing agarose gel electrophoresis to verify 28S and 18S rRNA content as indicators for ribosome distribution (<i>lower panel</i>).</p

CM induces cyp24a1 translation.

<p>MCF7 cells were treated with Ctr or CM for 4(A) and cyp24a1 (B) was analyzed in single fractions using RT-qPCR. The distribution of the respective mRNAs across the individual gradients was determined relative to the total RNA extracted from the gradients. Results from a representative experiment are given in A and B. (C+D) Changes of gapdh (C) and cyp24a1 (D) mRNA distribution induced by CM were normalized to Ctr. (E) cyp24a1 distribution (from D) was normalized to gapdh distribution (from C). Distribution changes are presented as means ± SEM (n≥3, * p<0.05, ** p<0.01, *** p<0.001).</p

Cyp24a1 translation is initiated in part cap-independently.

<p>MCF7 cells were treated with rapamycin [100 nM] for 4 h and subjected to polysomal fractionation. RNA from single fractions was isolated and gapdh (A) and cyp24a1 (B) mRNA distribution changes were analyzed separately as described before. Data are presented as means ± SEM (n≥3).</p

CM induces cyp24a1 IRES activity in an Akt-dependent manner.

<p>(A) MCF7 cells were transfected with phpR-cyp-F. 48 h after transfection cells were treated for 4 h with Ctr, CM, or CM supplemented with LY294002 [10 µM] or SB203580 [10 µM]. IRES activity was calculated as ratio of <i>firefly</i> to <i>renilla</i> luciferase activities and is given relative to Ctr. Data are presented as means ± SEM (n≥3, * p<0.05). (B) <i>(upper panel)</i> HEK293 cells overexpressing HA-tagged myr Akt were transfected with phpR-cyp-F. 48 h after transfection IRES activity was calculated as ratio of <i>firefly</i> to <i>renilla</i> luciferase activities and is given relative to control vector transfected cells. Data are presented as means ± SEM (n≥3, * p<0.05). <i>(lower panel)</i> HEK293 cells stably overexpressing HA-tagged myr Akt were serum starved for 48 h. Protein expression and S6-phosphorylation was determined by Western analysis. (C) MCF7 cells were treated for 4 h with CM or CM in combination with LY294002 [10 µM] followed by polysomal fractionation. Changes in cyp24a1 mRNA distribution were analyzed as described before. Data of pooled polysomal fractions (7–10) are presented as means ± SEM (n≥3, * p<0.05).</p

Erioflorin stabilizes Pdcd4_(39–91)luc from TPA-induced degradation.

<p>(A) Structure of erioflorin (Mwt = 348.4). (B) Stably Pdcd4_(39–91)luc expressing HEK293 cells were treated for 8 h with TPA (10 nM) with increasing concentrations of erioflorin (0.0625–10 µM). Pdcd4 stabilizing activity was determined relative to Δ(RLU_control–RLU_TPA-only). (C) Stably Pdcd4_(mut39–91)luc expressing HEK293 cells were treated as in (B). Luciferase activity is given relative to TPA-treated controls. All data are presented as means ± SEM (n≥3, *p<0.05, **p<0.01).</p

Erioflorin stabilizes Pdcd4 without affecting phosphorylation events.

<p>(A + B) HEK293 cells were transiently transfected with Pdcd4_(39–91)luc (A) or Pdcd4_(mut39–91)luc (B) <i>firefly</i> reporter vectors, in combination with expression vectors for either wildtype (S6K_wt = white bars) or constitutively active p70^S6K (S6K_ca = black bars) and a <i>renilla</i> luciferase vector one day prior to the experiment. Transfected cells were treated for 8 h with rapamycin (100 nM) or erioflorin (5 µM). <i>Firefly</i> normalized to <i>renilla</i> 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).</p

Erioflorin inhibits AP-1- and NF-κB-trancriptional activities.

<p>(A) HEK293 cells were co-transfected with an AP-1 <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TPA (10 nM) with or without erioflorin (2.5 and 5 µM). Relative AP-1 activity was normalized to <i>renilla</i> luciferase and presented relative to TPA-only treated cells. (B) HEK293 cells were co-transfected with a NF-κB <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TNFα (20 mg mL^–1) with or without erioflorin (2.5 and 5 µM). Relative NF-κB activity was normalized to <i>renilla</i> luciferase and presented relative to TNFα-only treated cells. All data are given as means ± SEM (n≥3, **p<0.01).</p

Erioflorin specifically inhibits E3-ligase β-TrCP1 activity.

<p>(A) HEK293 cells were treated with TNFα (20 ng mL^–1) for the indicated times with or without erioflorin (10 µM). (B) HEK293 cells were maintained under full medium conditions (10% serum) or serum deprived for 24 h following treatment with erioflorin (5 µM) for 8 h. (C) HEK293 cells were treated for 8 h with erioflorin (1.25–10 µM) or the prolyl-hydroxylase inhibitor dimethyloxallylglycine (DMOG, 1 µM). (D) HeLa cells were serum deprived for 48 h prior to treatment with erioflorin (10 µM) or the proteasome inhibitor MG132 (10 µM) for 8 h. Whole cell extracts were subjected to western analysis and probed with the indicated antibodies. Blots are representative for at least three independent experiments.</p

Erioflorin inhibits cell proliferation and alters cell cycle progression.

<p>(A, C, E) MCF7, HeLa and RKO cells were seeded at 10–20% confluency one day prior to the experiment and treated with DMSO (black diamonds) or erioflorin (2.5 (white triangles) and 5 µM (gray squares)). Cell confluency was followed for six days. (B, D, F) MCF7, HeLa and RKO cells were serum deprived for 48 h and treated with erioflorin (5 µM) for 16 h. After propidium iodide staining, distribution of the cells to the different phases of the cell cycle (subG1 (white), G1 (light gray), S (dark gray), G2/M (black)) was determined. (G) RKO colon carcinoma cells were subjected to a scratch wound assay. After administration of the scratch, medium was changed to control or erioflorin (5 µM) containing medium. Wound closure was measured after 24 h and relative wound closure is given as the ratio of the width of the scratch at 24 h and to 0 h. All data are given as means ± SEM (n≥3, *p<0.05, **p<0.01, ***p<0.001).</p