Beclin 1+/− has no effect on cell death, proliferation and ROS levels in thymocytes.

<p><b>A</b>) Isolated thymocytes from mice of the indicated genotypes were treated with Dex (1μM), using Q-VD-OPh (50 μM) as positive control for Caspase inhibition. Viability was determined over time. The means ± SD of at least 2 mice per group are shown. B) DNA content analysis of thymocytes (top panel) and thymic cellularity (lower panel) were performed on freshly isolated thymocytes from mice of the indicated genotypes between 8 and 12 weeks of age. Each symbol corresponds to the percentage of cycling cells (%S/G2/M) and the total viable thymocytes observed from an individual mouse. The mean ± SD of each group is shown. C) Representative histograms overlays of DHE and MitoSOX Red staining of the viable thymocytes isolated from Beclin 1+/+ (gray shaded) and Beclin 1+/− (black line) mice following the indicated treatment are shown. The viability of thymocytes following 24h of Dex treatment: 7.3% (Beclin 1+/+) and 8.9% (Beclin 1+/−). These results are representative of at least 3 independent experiments. D) Representative histogram overlays of the DHE and MitoSOX Red staining of the viable thymocytes isolated from Beclin 1+/+ and Beclin 1+/− mice are shown. For each graph, Beclin 1+/+ cells without caspase inhibition (gray shaded), Beclin 1+/+ cells with caspase inhibition (black line) and Beclin 1+/− cells with caspase inhibition (green line) either by Casp9DN expression or Q-VD-OPh treatment are shown. The viability of thymocytes following 24h of Dex treatment was as follows: 7.3% (Beclin 1+/+), 20% (Beclin 1+/+ x Casp9DN), and 22% (Beclin 1+/− x Casp9DN), 42% (Beclin 1+/+ Q-VD-OPh), and 43% (Beclin 1+/− Q-VD-OPh).</p

Casp9DN expression has no effect on thymic cellularity and proliferation in Lck-Bax38/1 mice.

<p><b>A</b>) DNA content analysis was performed on freshly isolated thymocytes from mice of the indicated genotypes between 8 and 10 weeks of age. Each symbol corresponds to the percentage of cycling cells (%S/G2/M) observed from an individual mouse. The mean ± SD of at least 6 mice per group is shown. <b>B</b>) Total thymic cellularity from mice of the indicated genotypes between 8 and 10 weeks of age is shown. Each symbol corresponds to the total viable thymocytes number observed from an individual mouse. The mean ± SD of at least 6 mice per group is shown.</p

Casp9DN expression does not promote lymphoma formation in Lck-Bax38/1 transgenic mice.

<p>Kaplan-Meier analysis for lymphoma free survival <b>A</b>) comparing wild type (black open square, N = 8) and Lck-Casp9DN (green solid triangle, N = 12) mice, and <b>B</b>) comparing Lck-Bax38/1 (black open square, N = 12) and Lck-Casp9DN x Lck-Bax38/1 (red solid triangle, N = 11) mice. Those mice were followed for lymphoma-free survival as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019786#s4" target="_blank">materials and methods</a>. P-value of analysis is shown. ND = not determinable without any events in the control group.</p

Casp9DN reduces ROS levels in primary thymocytes.

<p><b>A</b>) Representative histogram overlays of DHE stained viable thymocytes is shown. All cells were treated with or without Dex as indicated. Caspase activity was inhibited by either Casp9DN expression (red line, Casp9DN) or Q-VD-OPh treatment (blue line, WT+QVD) and compared to control cells (gray shaded, WT-wild type thymocytes). For the histogram overlay graphs, the viability of thymocytes was as follows; 24 h culture without Dex: 46% (WT), 56% (Casp9DN), and 69% (WT+QVD); 24 h culture with 1μM Dex: 7.5% (WT), 20% (Casp9DN), and 42% (WT+QVD). Quantitation of the percentage of cells with low DHE staining (gate shown in histograms) from the samples as indicated is shown in the bottom panel. The mean + SD of at least duplicate mice per group is shown. § p<0.05 versus the control. * p<0.01 versus the control. B) Representative histogram overlays of the MitoSOX Red stained viable thymocytes as indicated is shown. Data is representative of at least 3 independent animals. C) Representative histogram overlays of the DHE staining of the viable freshly isolated thymocytes from wild type mice (gray shaded, WT) and Lck-Casp9DN mice (red line, C9DN) is shown. The numbers are the percentage of cells found within the indicated gate for each sample. Quantitation of % DHE-Low population in freshly isolated thymocytes from mice of the indicated genotypes is shown. The mean + SD of at least 4 mice per group is shown.* p<0.005.</p

Beclin 1+/− does not accelerate lymphoma formation.

<p><b>A</b>) Primary thymocytes were isolated from mice of the indicated genotypes. Immunoblots for Beclin 1, and actin as a loading control are shown. The ratio of Beclin 1 to actin for each mouse is shown below the immunoblots. Kaplan-Meier analysis for lymphoma free survival <b>B</b>) comparing Beclin 1+/+ x Lck-Casp9DN (black open square, N = 12) and Beclin 1+/− x Lck-Casp9DN (green solid diamond, N = 14) mice, <b>C</b>) comparing Beclin 1+/+ x Lck-Bax38/1 (black open square, N = 11) and Beclin 1+/− x Lck-Bax38/1 (green solid diamond, N = 13) mice, and <b>D</b>) comparing Beclin 1+/+ x Lck-Bax1 (black open square, N = 15) and Beclin 1+/− x Lck-Bax1 (green solid diamond, N = 12) mice is shown. Those mice were followed for lymphoma-free survival as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019786#s4" target="_blank">materials and methods</a>. P-value of analysis is shown.</p

Caspase inhibition increases abnormal mitochondria and activates mitophagy.

<p><b>A</b>) TEM images showed morphologic changes in mitochondrial structures (normal mitochondria marked with black arrows and abnormal mitochondria marked with yellow arrows) in isolated thymocytes from mice of the indicated genotypes following the indicated treatment. One of examples of mitophagy showed that a mitochondrial cluster was engulfed within double-membrane autophagosome in bottom-right image. Four images have the same magnification. Scale Bar  = 0.2 µm. N = nucleus. <b>B</b>) Quantitation of the average number of normal and abnormal mitochondria per viable cell (left panel) and the percentages of cells with at least 1 normal mitochondrion or with at least 1 abnormal mitochondrion (right panel) in isolated thymocytes from mice of indicated genotypes following 4 h of the indicated treatment. <b>C</b>) Quantitation of the percentage of cells with at least 1 abnormal mitochondria in isolated thymocytes from mice of the indicated genotypes following various times of Dex treatment. Table shows that no abnormal mitochondria were observed in WT thymocytes following various treatments. In the WT thymocytes treated with 24 h of Dex, 5% of the cells were viable, so this sample is not shown (@). WT thymocytes treated with 0 h of Q-VD-OPh were not available (NA). <b>D</b>) In FL5-Neo cells, quantitation of the average number of normal and abnormal mitochondria per viable cell (left panel) and the percentages of viable cells with at least 1 normal mitochondrion or with at least 1 abnormal mitochondrion (right panel) following 24 h of the indicated treatments. <b>E</b>) FL5-Neo cells transfected with LC3-GFP (green) were stained with MitoTracker Red (red, MR) for mitochondria and TO-PRO-3 probe (blue) for nuclear counter staining following 48 h of the indicated treatment. The viability of FL5 cells: 97% (+IL-3), 9% (-IL-3), and 66% (-IL-3+QVD). Colocalization of MR and LC3-GFP indicated mitophagy activation. <b>F</b>) FL5 cells with the indicated expression were immuno-stained with p62 (green), MR (red) for mitochondria and TO-PRO-3 probe (blue) for nuclear counter staining following 48 h of IL-3 deprivation and BafA (20 nM). Pearson's correlation coefficient (PCC, R(r)) which statistically measures colocalization of p62 and MR is shown in each image. Quantitation of PCC of the treated FL5 cells is shown. Each symbol corresponds to PCC of each viable cell of the indicated cell lines. The mean ± SD of at least 22 cells per cell line in each treatment is shown. ** p<0.0001.</p

Quantitative Analysis and Discovery of Lysine and Arginine Modifications

Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation

A WEE1 Inhibitor Analog of AZD1775 Maintains Synergy with Cisplatin and Demonstrates Reduced Single-Agent Cytotoxicity in Medulloblastoma Cells

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure–activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin