The effect of knock-down RelB on radioresistance of PC3 tumors.

<p>A lentivirus-based RelB siRNA knock-down PC3 clone was characterized by Western blots (A). The RelB shRNA/PC3 cells were injected into male mice for tumor formation (B). The tumors were treated with IR (3×5 Gy) when tumor volumes reached 500 mm³ and tumors were allowed to grow to 2000 mm³ (C). RelB and IL-8 levels were quantified in the tumor lysates and statistically analyzed (D). Statistical analysis for tumor volumes and the levels of target proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032905#pone-0032905-g004" target="_blank">Figure 4</a>.</p

Reverse correlation of PSA and IL-8 in PCa cells.

<p>To manipulate PSA and IL-8 in PCa cells, LNCaP cells were transfected with an IL-8 cDNA construct and a PSA siRNA (A); PC3 cells were transfected with PSA cDNA construct and IL-8 siRNA (B). To test whether PSA influences NF-κB transcriptional activation, a NF-κB-luciferase reporter construct was co-transfected with a PSA expression construct or PSA siRNA and a β-gal expression construct into LNCaP cells. The luciferase responses were normalized with β-gal activities (C). PSA and IL-8 levels were quantified by Western blots with normalization as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032905#pone-0032905-g001" target="_blank">Figure 1</a>.</p

The effects of PSA and IL-8 on radioresistance of PCa cells.

<p>To determine the roles of PSA and IL-8 in radiosensitivity of PCa cells, LNCaP cells were treated with IL-8 protein or stably transfected with IL-8 cDNA (A) and stably transducted with PSA shRNA lentivirus (B); PC3 cells were stably transducted with PSA cDNA and IL-8 shRNA lentivirus (C). The levels of PSA were quantified by Western blots and the levels of IL-8 were quantified by either Western blots or an Elisa kit. Cell survival was quantified by Trypan blue exclusion assays. Western blots were normalized with controls and cell survival fraction analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032905#pone-0032905-g001" target="_blank">Figure 1</a>.</p

The effect of overexpression of RelB on radioresistance of LNCaP tumors.

<p>A stable RelB expressed LNCaP clone was characterized by Western blots (A). The RelB/LNCaP cells were injected into male mice for tumor formation and growth reaching to 500 mm³ (B). The tumors were treated IR (5×3 Gy) and tumor volumes were measured until they reached 2000 mm³ (C). RelB, PSA and IL-8 levels were quantified in the tumor lysates and statistically analyzed (D). Statistical significances of tumor volumes (C) and the levels of the target proteins (D) between RelB expressed and vehicle control in both no- IR and IR treated groups are indicated.</p

Suppression of tumorigenicity of PC-3 by expression of miR-17*.

<p>A, has-miR-17* was cloned in a Tet-on lentiviral vector and stably transected into PC-3 cells. The clone was tested by RFP screening under Dox- inductive conditions and then confirmed by measuring the expression of the three target genes using Western blots with β-actin normalization. B and C, the generated clone was injected into male nude mice to determine its tumorigenicity. The vehicle control was included. The number of days needed for tumor size to reach 500 mm³ is shown in (B) and calculated tumor growth rates in (C). D, total RNA and proteins were isolated from the tumor tissues and the level of miR-17* and corresponding activities of the three antioxidant proteins were quantified. Three samples (n = 3) were used in testing the generated miR-17* inducible clone (A). Nine vehicle control animals (n = 9) with or without DOX treatment and eighteen miR-17* expressed animals (n = 18) with or without DOX treatment were used to test the effect of miR-17* on tumor growth (B), (C), (D). * (p<0.05) and ** (p<0.01) indicate significances as compared to without DOX control (A) and (C).</p

Identification of three mitochondrial antioxidant proteins as miR-17* targets.

<p>A, the levels of miR-17 and miR-17* expressed in PCa and control cell lines were measured by RT-PCR. The ratio of miR-17 to miR-17* in each cell line is presented. B and C, transfection of miR-17* in PC-3 cells to validate its function in repressing the expression of antioxidant proteins and diminishing TNF-mediated MnSOD induction. D, the repressive effect of miR-17* on the antioxidant proteins is estimated by quantitative luciferase reporter assay. RNU24 and β-actin were used as internal controls to normalize miRNA levels (A), protein levels (B and C). Images were normalized with the internal controls and then normalized by PrEC (A), by control miRNA (B), and by no TNF treatment (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments and fold changes in Western blots are indicated. * (p<0.05) and ** (p<0.01) indicate significances as compared to the controls: PrEC (A), control miRNA (B) and (D), and untreated samples (C).</p

Cytotoxicity of miR-17* in PCa cells.

<p>A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).</p

Induction of miR-17* in PCa cells by DSF.

<p>A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).</p

Global Effects of Adriamycin Treatment on Mouse Splenic Protein Levels

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and “chemobrain”, which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC–MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca²⁺ binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics

NFκB DNA binding activity is increased in WT mice.

<p>Nuclear extract (12 µg) from heart tissue of WT, iNOS (−/−), TgM (++), and iNOS (−/−)-TgM (++) mice was isolated and assayed for NFκB DNA binding activity. Densitometric quantitation as fold increase from respective saline control is shown (with gel shift inset). *p<0.001 versus saline for WT.</p