Additional file 3: Figure S2. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

The effect of PD98059 inhibitor on TGF-β induced nuclear accumulation of Smad2/Smad3 complex. (PDF 352 kb

Additional file 4: Figure S3. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

The effect of AGR2 expression on vimentin cellular localization. A scale bars correspond to 20 μm. (PDF 319 kb

Additional file 2: Table S1. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

Sequences of the primers used in quantitative PCR analysis. (PDF 213 kb

Titanocene Dihalides and Ferrocenes Bearing a Pendant α‑d‑Xylofuranos-5-yl or α‑d‑Ribofuranos-5-yl Moiety. Synthesis, Characterization, and Cytotoxic Activity

Titanocene dichlorides of general formula [(η⁵-C₅H₅)­(η⁵-C₅H₄R)­TiCl₂] (where R = 5-deoxy-1,2-di-<i>O</i>-isopropylidene-3-<i>O</i>-benzyl-α-d-xylofuranos-5-yl (Xylf) (<b>8a</b>); R = 5-deoxy-1,2-di-<i>O</i>-isopropylidene-3-<i>O</i>-benzyl-α-d-ribofuranos-5-yl (Ribf) (<b>8b</b>)) and [(η⁵-C₅H₄R)₂TiCl₂] (R = Xylf (<b>9a</b>); R = Ribf (<b>9b</b>)) were prepared by reaction of the corresponding lithium cyclopentadienides <b>7a</b>,<b>b</b> with an equimolar amount of [(η⁵-C₅H₅)­TiCl₃] or a 0.5 mol amount of [TiCl₄(THF)₂]. Titanocene difluorides of the general formula [(η⁵-C₅H₄R¹)­(η⁵-C₅H₄R²)­TiF₂] (R¹ = H and R² = Ribf (<b>10</b>); R¹ = R² = Xylf (<b>11a</b>); R¹ = R² = Ribf (<b>11b</b>)) were obtained by fluorination of the corresponding titanocene dichlorides <b>8b</b> and <b>9</b> with the fluorinating agent {2-(CH₂NMe₂)­C₆H₄-κ<i>C</i>,<i>N</i>}­(<i>n</i>-Bu)₂SnF in high yields. Alternatively, complexes <b>11</b> were prepared in a straightforward way by direct reaction of [TiF₄(THF)₂] with 2 equiv of the corresponding lithium cyclopentadienide <b>7a</b>,<b>b</b>. Ferrocene complexes [(η⁵-C₅H₄R)₂Fe] (R = Xylf (<b>12a</b>); R = Ribf (<b>12b</b>)) were synthesized by metathesis of 2 equiv of lithium cyclopentadienide <b>7a</b>,<b>b</b> and 1 equiv of anhydrous FeCl₂. Deprotection of the benzyl group in ferrocenes <b>12</b> proceeded cleanly by a catalytic hydrogenation on Pd/C and afforded the ferrocene diols [(η⁵-C₅H₄R)₂Fe] (R = 5-deoxy-1,2-di-<i>O</i>-isopropylidene-α-d-xylofuranos-5-yl (Xylf-OH) (<b>14a</b>); R = 5-deoxy-1,2-di-<i>O</i>-isopropylidene-α-d-ribofuranos-5-yl (Ribf-OH) (<b>14b</b>)). A scaled up benzyl deprotection with Et₃SiH as a hydrogen source led to the replacement of only one benzyl group, which gave the ferrocene alcohol [(η⁵-C₅H₄R¹)­(η⁵-C₅H₄R²)­Fe] (R¹ = Xylf and R² = Xylf-OH (<b>13</b>)). The prepared complexes were characterized by elemental analysis, melting point determination, NMR, IR, and ESI-MS, and the molecular structure of <b>9b</b> was determined by X-ray diffraction analysis. The cytotoxic activity of complexes <b>8</b>–<b>14</b> against A2780 and A2780cis cancer cells was evaluated by MTT tests. Titanocene difluorides <b>10</b> and <b>11</b> and ferrocene diol <b>14a</b> showed cytotoxicity against A2780 cells in the medium to low micromolar range, while the most active species, <b>11b</b>, displayed about 40% higher cytotoxicity against A2780cis in comparison to a cisplatin standard

The experimental setup:

<p>1. Total RNA was extracted. 2. Reverse transcription was performed in two different setups i) using random hexamers to collect all possible types of RNA transcripts and ii) using oligo dT primers to gain polyadenylated types of RNA transcripts. 3) Real-time PCR with primers designed to specifically recognize N- and C-terminus of β-galactosidase cDNA was used to amplify sequences at both 5′-end (PCR-1) and 3′-end (PCR-2) of β-galactosidase RNA transcripts.</p

The effect of OCII and Rosc on phosphorylation of RNA polymerase II CTD.

<p>(A) Phosphorylation of RNA polymerase II CTD in wild type p53 cells either with stably integrated SV40 (COS-1) or SV40-negative cells (CV-1) was determined. The phosphorylation of both Ser 2 and Ser 5 was inhibited after 12 h treatment either with 8 µM OCII or 20 µM Rosc in all tested cell lines. (B) Comparison of RNA polymerase II CTD phosphorylation in cells expressing Tat with pHIV-lacZ stably integrated into cellular genome (H1299-HIV) or with pHIV-lacZ as a part of extrachromosomal DNA (H1299-Tat). The phosphorylation of Ser 2 and Ser 5 in both cell lines was inhibited after PCI treatment for 12 h. The numbers represent the fold changes of samples to untreated controls.</p

OCII and Rosc suppress expression from extrachromosomal genetic elements.

<p>(A) MCF-7 cells transfected with pSV-lacZ were treated either with OCII (8 µM) or Rosc (20 µM). Relative levels of β-galactosidase activity were determined in a chromogenic (ONPG) enzyme assay. The Y axis represent<b>s</b> relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates. (B) OCII and Rosc induce wild type p53-mediated transactivation of p21^WAF−1. MCF-7 (wild type p53) cell line was treated either with OCII (8 µM) or Rosc (20 µM) for 24 h and analyzed for the expression of p53, p21^WAF−1 and actin by immunoblotting. The numbers represent the fold changes of samples to untreated controls.</p

The effect of PCIs on the integrity of synthesized RNA.

<p>We compared amounts of total and full length polyadenylated transcripts of β-galactosidase gene after treatments with OLII and Rosc. We quantified total and full length transcripts of β-galactosidase gene in cell lines H1299-HIV (B) and H1299-Tat transiently transfected by pHIV-LacZ (A) by using qRT-PCR. PCIs stimulated the RNA synthesis from viral promoters ((B) H1299-HIV PCR-1, Random hexamers (A) H1299-Tat, PCR-1, Random hexamers) however viral promoters incorporated in the cellular genome generated higher amounts of full length RNA transcripts ((B) H1299-HIV, PCR-2, OLIGO dT). The amount of full length polyadenylated transcripts produced from extrachomosomal viral promoters after treatments with PCIs were significantly lower ((A) H1299-Tat, PCR-2, OLIGO dT). Data were determined by relative quantification with control PCR-1 and control PCR-2 as the calibrators. The Y axes represent the fold change of β-galactosidase RNA transcripts after PCIs treatment to the amount of β-galactosidase RNA transcripts in controls. The error bars illustrate the standard deviation of three independent biological replicates.</p

Tamoxifen-Dependent Induction of <i>AGR2</i> Is Associated with Increased Aggressiveness of Endometrial Cancer Cells

<p>Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher <i>AGR2</i> expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated <i>AGR2</i> level with myometrial invasion occurrence and invasion depth was also found. <i>In vitro</i> analyses identified a stimulatory effect of <i>AGR2</i> on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated <i>AGR2</i> as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.</p

Inhibition of expression from extrachromosomal HIV promoter by PCI.

<p>A β-galactosidase reporter assay was used to measure expression from the promoter within the pHIV-lacZ plasmid following transfection into a Tat-expressing cell line (H1299-Tat). Results were compared with expression from Tat-expressing cell line with pHIV-lacZ stably integrated into the cellular genome (H1299-HIV). The requirement for Tat to promote expression from HIV promoter was illustrated in H1299 (Tat-negative) control cells transiently transfected with pHIV-lacZ. The Y axis represents relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates.</p