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

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

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

OCII and Rosc suppress T-antigen expression.

<p>(A) OCII and Rosc suppress T-antigen expression and restore p53–mediated transactivation of p21^WAF−1 during SV40 infection. MCF-7 (wild type p53) cell line infected with SV40 was treated either with 8 µM OCII or 20 µM Rosc, either immediately or 16 h post-infection. (OCII 16 h, Rosc 16 h). Infected cells were harvested at 45 h and then analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. (B) Inhibition of expression from viral promoter integrated into cellular genome. Cell lines CV-1 and COS-1 were treated either with 8 µM OCII or 20 µM Rosc and analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. CV-1 cell line was used as SV40-negative control cell line (no large T-antigen expression). The numbers represent the fold changes of samples to untreated controls.</p

Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.

<p>Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.</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

Additional file 3: of ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

List of genes whose expression was changed 24 h after induction of ∆Np63α or TAp63α expression in MDA-MB-468 cells. (PDF 503 kb

Additional file 3: of ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

List of genes whose expression was changed 24 h after induction of ∆Np63α or TAp63α expression in MDA-MB-468 cells. (PDF 503 kb

Quantitative Proteomic Profiling of Pleomorphic Human Sarcoma Identifies CLIC1 as a Dominant Pro-Oncogenic Receptor Expressed in Diverse Sarcoma Types

Sarcomas are rare forms of cancer with a high unmet clinical need that develop in connective tissue, such as muscle, bone, nerves, cartilage, and fat. The outcome for patients is poor, with surgery and postoperative radiotherapy the standard treatment for patients. A better understanding of the molecular pathology of sarcoma may allow for the development of novel therapeutics. There are dozens of sarcoma subtypes where there is a need for targetted therapeutics, with the most commonly studied including Ewing’s sarcoma and osteosarcoma. Here we initiate a proteomics-based target-discovery program to define “dominant” pro-oncogenic signaling targets in the most common sarcoma in adults: high-grade pleiomorphic soft tissue sarcoma. We have carried out a proteome screen using tandem mass tag isobaric labeling on three high-grade undifferentiated pleomorphic sarcoma biopsies from different tissue sites. We identified the commonly dysregulated proteins within the three sarcomas and further validated the most penetrant receptor as CLIC1, using immunohistochemistry arising from two different population cohorts representing over 300 patients. The dominant expression of CLIC1 in a broad range of human sarcomas suggests that studying this relatively unexplored signaling pathway might provide new insights into disease mechanism and facilitate the development of new CLIC1 targeted therapeutics