Effect of low temperature and DMSO on PC biosynthesis.

<p>CHO-K1 cells stably expressing PC-wt or PC-A267T were incubated at 26°C (A) or in culture medium containing 2% DMSO (B). PC concentrations were quantified in cell lysates (▪) and culture medium (□), and normalized to total protein (TP) concentrations of the corresponding lysate samples. Histograms and the bars represent mean + SEM values, n = 9. * (p≤0.05, Mann-Whithey test), PC-wt/PC-A267T treated versus PC-wt/PC-A267T untreated.</p

Expression levels of ER stress and UPR activation markers.

<p>Immunoglobulin-binding protein (BiP) (A) and phosphorylated eukaryotic initiation factor 2α (P-eIF2α) (B) levels were analyzed by Western blotting in lysates from CHO-K1 cells stably expressing PC-wt (lane 1) or PC-A267T (lane 2). Fold increase of BiP and P-eIF2α levels in CHO-K1 cells expressing PC-A267T are shown as histograms and bars, which represent mean + SEM values of expression levels detected in three independent experiments. BiP bands were quantified and normalized to corresponding amounts of α-tubulin. P-eIF2α bands were quantified and normalized to corresponding amounts of eIF2α.</p

DNA fragmentation analysis in CHO-K1 cells stably expressing PC-wt or PC-A267T.

<p>The levels of cytoplasmic DNA fragments (mono- and oligonucleosomes) in cells stably expressing PC variants was measured by the Cell Death Detection ELISA^PLUS kit. The results were normalized to total protein (TP) concentrations and presented as mean + SEM values of four independent experiments (each experiment performed in triplicates). * (p≤0.05, Mann-Whithey test).</p

Identification of PC-chaperone complexes in CHO-K1 cells stably expressing PC-wt or PC-A267T.

<p>The cells were pretreated with dithiobis[succinimydylpropionate] (DSP) before cell lysis. Cell lysates with equivalent amounts of PC were separated on SDS-PAGE under reducing conditions and immunoblotted with anti-PC to detect PC-chaperone complexes. Similar results were obtained in three independent experiments. The molecular weight standards are shown on the right (kDa) and the positions of the PC-chaperone complexes are marked with the arrows on the left (A). Identification of chaperones associated with the PC mutant. The cell lysates pretreated with DSP were subjected to SDS-PAGE and Western blot analysis with antibodies against indicated chaperones. Two independent experiments with similar results were performed. (B).</p

TFPI Alpha and Beta Regulate mRNAs and microRNAs Involved in Cancer Biology and in the Immune System in Breast Cancer Cells

<div><p>Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, possibly by signal transduction involving the EGFR pathway.</p> </div

Validation of microarray and miRNA array expression results.

<p>Selected mRNAs (A and B) and miRNAs (C and D) differentially expressed in SK-BR-3 cells overexpressing TFPIα (A and C) or TFPIβ (B and D) were validated by Taqman single assays and qRT-PCR. Results were normalized against the endogenous controls PMM1 and U6 snRNA and the relative expression calculated using the comparative Ct method. Values are presented as mean (n = 3) fold change (FC) of empty vector pTOPO control + SEM of three biological replicates. White bars indicate array expression values, black bars represent Taqman verification values. Dotted lines indicate FC  = |2|.</p

Transcription factor activity and phospho-Akt in cells after overexpression of TFPI.

<p>(A) Transcription factor activity was measured using the cignal finder luciferase reporter system. SK-BR-3 cells (3×10⁴) transiently transfected with vectors overexpressing TFPIα (dark gray) or TFPIβ (gray), or with empty vector pTOPO (light gray) as controls were seeded in 96-well arrays 24 hours after transfection. After 48 hours, cells were lysed and the firefly and renilla luciferase intensity determined. The results are presented as mean (n≥8) relative luciferase activity + SEM of three independent experiments. Statistical differences between cells overexpressing TFPI and empty vector control cells were determined using the student's t test (* <i>p</i><.05, ** <i>p</i><.01, *** <i>p</i><.001, ns  =  not significant). (B) Western blot of phospho-Akt in SK-BR-3 cells overexpressing TFPI. Band intensities were measured using ImageJ and the intensities of phospho-Akt were normalized to Akt levels.</p

Venn diagrams of differentially expressed miRNAs after overexpression of TFPIα or TFPIβ.

<p>miRNAs associated with molecular and cellular function (A) and cancer disease (B), as annotated by the Ingenuity Pathway Analysis software.</p

Clinical relevance of gene signatures following overexpression of TFPIα or TFPIβ.

<p>Three publicly available, clinically annotated breast cancer datasets (GSE6532, GSE4922, and GSE7390) were downloaded from the Gene Expression Omnibus (GEO) database at NCBI and merged. Associations between the differentially expressed genes and clinical variables were evaluated using the <i>globaltest</i> package in R.</p

EGFR protein levels detected in the lysate of transfected SK-BR-3 cells by Western blot analysis.

<p>Proteins were separated on a SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and detected using an anti-EGFR antibody. Anti-actin was used as a protein loading control. (A) Western blot of one representative experiment. (B) Quantification of three independent experiments using ImageJ (n = 3+ SEM).</p