Additional file 3: of V-J combinations of T-cell receptor predict responses to erythropoietin in end-stage renal disease patients

Summary of repertoire sequencing output and overlapping proportions in end-stage renal disease samples. (DOCX 14 kb

Additional file 2: of V-J combinations of T-cell receptor predict responses to erythropoietin in end-stage renal disease patients

Overlapping read length distribution plot of end-stage renal disease patients. QC, quality control. (DOCX 260 kb

Additional file 7: of V-J combinations of T-cell receptor predict responses to erythropoietin in end-stage renal disease patients

Quantification of T-cell repertoire diversity in end-stage renal disease (ESRD) patients. a The clonotype distribution plots of ESRD patients. The x-axis and y-axis are log10 scaled. The value of 1/D represents the T-cell receptor (TCR) β repertoire diversity. b Comparison of TCR repertoire diversity (log-scaled) between erythropoietin (EPO) responders and EPO resistants (p = 0.642) (DOCX 232 kb

Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

<div><p>The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR) are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65) was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.</p> </div

Loss of ZBRK1 Contributes to the Increase of KAP1 and Promotes KAP1-Mediated Metastasis and Invasion in Cervical Cancer

<div><p>ZBRK1, a zinc finger protein that interacts with breast cancer 1 (BRCA1) and KRAB-ZFP-associated protein 1 (KAP1), has been suggested to serve as a tumor suppressor via repression of tumor metastasis/invasion. To date, the detailed molecular mechanisms for how BRCA1 and KAP1 participate in ZBRK1-mediated transcriptional repression, metastasis and invasion as well as the associated clinical relevance remain unclear. In this study, we demonstrated that both the N- and C-terminal domains of ZBRK1 are important for inhibiting cell proliferation and anchorage-independent growth in cervical cancer. Specifically, the N-terminal KRAB domain of ZBRK1 displayed a more crucial role in inhibiting metastasis and invasion through modulation of KAP1 function in a transcriptionally dependent manner. The loss of ZBRK1 results in an increase of KAP1 expression, which enhanced migration and invasion of cervical cancer cells both the <i>in vitro</i> and <i>in vivo</i>. Moreover, an inverse correlation of expression levels was observed between ZBRK1 and KAP1 following tumor progression from in situ carcinoma to invasive/metastatic cervical cancer specimens. Taken together, the current results indicate that a loss of ZBRK1 contributes to the increased expression of KAP1, potentiating its role to enhance metastasis and invasion.</p> </div

EGF-induced IL-1β reduces cisplatin-induced cytotoxicity.

<p>(<b>A-C</b>) A431 cells were treated with 50 ng/ml EGF and 5 ng/ml IL-1β for 6 h and followed by treating cells with 20 µM cisplatin (CDDP) for 24 h. Cell number and viability were analyzed by using cell counter (<b>A</b>) and the CCK-8 assay (<b>B</b>), respectively. Lysates were prepared and subjected to Western blot with antibodies against caspase-3 and actin (<b>C</b>). (<b>D and E</b>) Cells were treated with 50 ng/ml EGF and 5 ng/ml IL-1β for 6 h and followed by treating cells with IL-1β antibodies and 20 µM cisplatin (CDDP) for 24 h. The apoptotic cells were examined by using Annexin V and PI staining in flow cytometric analysis. The apoptosis ratio was then calculated as shown in (<b>E</b>). Values are means ± S.E. of three determinations. n.s.: no significant difference. ***: P<0.005; **: P< 0.01.</p

Activation of NF-κB is essential for EGF-induced IL-1β expression.

<p>(<b>A</b>) A431 cells were treated with various concentration of LY294002 as indicated for 1 h and followed by treated with 50 ng/ml EGF for 10 min (lysates) or 3 h (RNA). Lysates and RNA were prepared for examining the phosphorylation of Akt on serine 473 and IL-1βmRNA, respectively by using Western blot analysis and RT-PCR. (<b>B and D</b>) A431 cells were treated with various concentrations of LY294002 or parthenolide as indicated for 1 h, and followed by treated with 50 ng/ml EGF for 3 h. The expression of IL-1β was analyzed by Real-time PCR. The induction fold of EGF-induced IL-1β expression was normalized by GAPDH. (<b>C</b>) A431 cells were treated with various concentrations of parthenolide as indicated for 1 h and followed by treated with 50 ng/ml EGF for 1 h (nuclear extracts) or 3 h (RNA). Nuclear extracts and RNA were prepared for examining the nuclear translocation of NF-κB and IL-1βmRNA, respectively by using Western blot analysis and RT-PCR. Values represent means ± S.E. of three determinations. n.s.: no significant difference. ***: P<0.005; **: P< 0.01; *: P<0.05.</p

EGF induces the expression of IL-1β.

<p>(<b>A</b>) A431 cells were treated with 50 ng/ml EGF for a period of time (1∼9 h) before the extraction of RNA. The expression of IL-1β, IL-8, IL-11 and GAPDH mRNA was analyzed by RT-PCR and examined in 2% agarose gel. (<b>B and C</b>) A431 cells were treated with 50 ng/ml EGF for a period of time (1∼9 h) and various concentrations of EGF (1∼100 ng/ml). Expression of IL-1β was analyzed by Real-time PCR. The induction fold of EGF-induced IL-1β expression was normalized by GAPDH. (<b>D</b>) A431 cells were treated with 50 ng/ml EGF for 6 and 9 h, and then the condition media were collected for analyzing the IL-1β protein by ELISA. Values represent means ± S.E. of three independent experiments. ***: P<0.005; **: P< 0.01; *: P<0.05. N.D.: non-detected; n.s.: no significant difference.</p

Effect of EGF on IL-1β mRNA stability and transcriptional activation.

<p>(<b>A and B</b>) A431 cells were treated with 50 ng/ml EGF for 3 h and then treated with 5 µg/ml actinomycin D for a period of time (1∼9 h). The expression of IL-1β and GAPDH mRNA was analyzed by RT-PCR and examined in 2% agarose gel. The expression of IL-1β mRNA was also analyzed by Real-time PCR. Relative levels of IL-1β were normalized by GADPH and the degradation rate was carried out by comparing to 0 h (as 100%). (<b>C</b>) A431 cells were transfected with 0.5 µg IL-1β promoter construct by lipofection and then treated with 50 ng/ml EGF for various time as indicated. The luciferase activities and protein concentrations were then determined and normalized. Values represent means ± S.E. of three determinations.</p

NF-κB binding site located on IL-1β promoter is essential for EGF-induced promoter activity.

<p>(<b>A</b>) Constructs of IL-1β promoter or mutation sequence of NF-κB binding site bearing luciferase gene were presented. (<b>B and C</b>) Nuclear extracts (NE) from A431 cells were treated with 20 µM LY294002 (LY) and 20 µM parthenolide (PTL) for 1 h, and followed by treating with 50 ng/ml EGF for 1 h. DNA affinity precipitation assay was performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055795#s2" target="_blank">Materials and Methods</a>”. Binding of NF-κB p65 to wild-type probes, wt was analyzed by Western blot. The mutant sequence, NF-κB mut was used to serve as a negative control for wild-type sequence. (<b>D</b>) Cells were transfected with 0.5 µg IL-1β promoter construct by lipofection and then treated with 20 µM LY294002 (LY) and 20 µM parthenolide (PTL) for 1 h, and followed by treating with 50 ng/ml EGF for 6 h. The luciferase activities and protein concentrations were then determined and normalized. (<b>E</b>) Cells were transfected with 0.5 µg wild type (wt) or mutant (NF-κB mut) IL-1β promoter construct by lipofection and then treated with 50 ng/ml EGF for 6 h. The luciferase activities and protein concentrations were then determined and normalized. Values of luciferase activities are means ± S.E. of three determinations. ***: P<0.005; n.s.: no significant difference; Ctrl: control.</p