Additional file 2: of BET protein inhibition regulates cytokine production and promotes neuroprotection after spinal cord injury

BET inhibition affects macrophage reactivity in vitro. Real-time PCR quantification of (A) pro-inflammatory and (B) anti-inflammatory cytokines at 3 h after LPS (100 ng/ml) stimulation, normalized to the GAPDH levels. Experiments were repeated 3 independent times. *p < 0.05, **p < 0.01, ***p< 0.005, as calculated by one-way ANOVA followed by Tukey post-hoc test. Data are represented as mean ± SEM fold changes of gene expression. (TIF 1378 kb

Additional file 1: of BET protein inhibition regulates cytokine production and promotes neuroprotection after spinal cord injury

JQ1 treatment modifies the M1 marker INOS but do not modify markers of M2 macrophage phenotype, or astrocyte reactivity. Real-time PCR quantification of (A) M1 and M2 markers and (B) macrophage and glial markers at 4 and 72 h after SCI, normalized to the GAPDH levels. N =3-4 mice/group. *p < 0.05, ***p < 0.005, as calculated by one-way ANOVA followed by Tukey post-hoc test. Data are represented as mean ± SEM fold changes of gene expression. (TIF 1771 kb

Screening of cell cycle fusion proteins to identify kinase signaling networks

<p>Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27^Kip1. We generated a p27^Kip1-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27^Kip1-luciferase fusion stabilization. This was combined with <i>in silico</i> kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27^Kip1 turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.</p

Additional file 3: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Figure S2. A longitudinal study on a single GBM patient was carried out in order to monitor the changes in serum HOTAIR expression over time. 3 different time points were included in this study: pre-op (the blood was drawn right before the surgery started), post-op (at least 24 h after surgery) and during the 2 week follow-up (F/U) with the neurosurgeon. We show that the level of HOTAIR decreases after surgery and at the follow-up visit. (PDF 430 kb

Additional file 1: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Supplementary materials and methods. (DOCX 109 kb

Additional file 2: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Figure S1. HOTAIR expression detected in our biomarker assay is derived mostly from circulating RNA, not DNA. 3 GBM serum samples were selected at random and the relative HOTAIR expression in GBM serum with and without reverse transcription (RT)-PCR was determined. The HOTAIR RNA was reverse-transcribed into HOTAIR cDNA and qPCR was performed. The circulating HOTAIR DNA in the serum was detected by qPCR without RT. The considerable difference between HOTAIR expression with and without RT demonstrates that the HOTAIR we are detecting in our qRT-PCR reactions is derived from RNA and not DNA. (PDF 1003 kb

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

<p>Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated. </p