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

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

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

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