developed to investigate LC3B lipidation . Finally, all samples were aliquoted and stored at −20 °C until they were used. The proteins were size-separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Western blots were performed according to the datasheets of the individual primary antibodies. Anti- bodies, including ACTB (#4970), ATG5 (#12994), BNIP3 (#3769), LC3B (#3868), MTOR (#2972), p-MTOR (#2971), p-RPS6 (#2211), RPS6 (#2217), and UB (#3936), were purchased from Cell SignalingTechnology. The antibodies against CTSE (sc-30055) and MIF (sc-20121) were purchased from Santa Cruz Biotechnology. The secondary antibodies for all primary antibodies were horse radish peroxidase (HRP)-conjugated (Cell Signal- ing Technology [#7074] for the primary antibodies from Cell SignalingTechnology, and secondary antibodies from Santa Cruz Biotechnology [sc-2030] were used for the primary antibodies from Santa Cruz Biotechnology. Subsequently, the binding of secondary antibodies was detected with HRP luminal substrate (Merck Millipore Corporation, WBKLS0100). The luminescence was mon- itored with Luminescent Image Analyzer ImageQuant LAS 4000 (GE Healthcare Life Sciences). The signal intensities were analyzed with ImageJ version 1.45 s soft- ware (NIH). ACTB was the internal loading control for all western blot experiments. Statistical significance was tested using a one-tailed t-test in Excel 2013 software (Microsoft).
using non-specific methods of visualization such as 32 P-orthophosphate metabolic labeling or pan-serine antibodies, as gain or loss of a single phospho-serine residue is squelched by background signal. In order to more specifically detect phosphorylation induced by IKKα we employed a phospho-serine motif antibody developed by Cell SignalingTechnology, Inc. (CST) for the purpose of screening proteins for partial 14-3-3 binding sites. This antibody, referred to hereafter as p-Ser (2981), recognizes an R-x-Y/F- x-pSer motif, which partially matches the IKK phosphorylation motif (see Figure 2.7A) , and is permissible for substitution of arginine with other hydrophilic amino acids at the -4 position (per conversations with CST technical staff). We confirmed that p-Ser (2981) recognized IKKα-phosphorylated AR, not non-phosphorylated AR, by
ies at 4°C to avoid light overnight. The primary antibodies AKT (p-AKT), GAPDH, and secondary antibody were purchased from Cell SignalingTechnology (USA), while P70S6K (p-P70S6K) were from Signalway Antibody LLC (USA), WWOX from ImmunoWay Biotechnology Company (USA). The next day, the PVDF membranes were placed in the fluorescent secondary antibody. Finally, sweeping instrument (LI-COR, USA) was used to detect specific proteins separated by electrophoresis.
ride (PVDF) filter membranes (Millipore, Bed- ford, MA, USA). After blocking with PBS contain- ing 5% non-fat milk and 0.1% Tween-20 for 2 hours, the membranes were then incubated with the primary antibodies overnight at 4°C. Then the membranes were washed three times with PBST for 5 minutes each time and incu- bated with horseradish peroxidase-conjugated secondary antibody for 2 h at room tempera- ture. Finally, the membranes were scanned by the ECL detection system. The band density was quantified using the Image J analysis sys- tem (Wayne Rasband, National Institutes of Health, USA). The primary antibodies includ- ed: anti-Gpx3 antibody (ab104448; 1:1000; Abcam), anti-Ecad antibody (14472; 1:1000; Cell SignalingTechnology), anti-Ncad antibody (D4R1H; 1:1000; Cell SignalingTechnology), anti-pβ-catinin antibody (D10A8; 1:1000; Cell
) and immunoprecipitated with Flag beads (Sigma-Aldrich A2220). Proteins were extracted with the same lysis buffer described above and subjected to SDS-PAGE elec- trophoresis. Protein extractions from the different tissues (eWAT, BAT, brain, muscle, heart, kidney, lung, spleen, and liver) were prepared using M-PER mammalian extraction buffer (Thermo Scientific) con- taining 1:100 Halt phosphatase inhibitor cocktail (Thermo Scientific) and 1:100 Halt protease inhibitor cocktail, EDTA-free (Thermo Sci- entific). All the tissues were snap-frozen and then ground with Liquid N2 before lysis. The following antibodies were used for Western blot analysis: anti-CCND1 (NeoMarkers Rb-010-P0), anti-CCND3 (clone sc-6283), anti-CDK4 (clone sc-260), anti-HSL (clone sc-25843), anti- HA (clone sc-805), anti-IRS2 (clone sc-8299) (Santa Cruz Biotechnol- ogy Inc.); anti-CCND2 (clone ab3085), anti-CDK4 (clone DSC-35), anti-Ki67 (clone ab15580) (Abcam); anti–LMNA (clone 2032), anti– pHSL Ser573 (clone 4139), anti–RB1 Ser780 (clone 9307), anti–pAKT Thr308 (clone 4056), anti–pAKT Ser473 (clone 4060), anti-AKT (clone 9272), anti-CDK6 (clone DCS83) (Cell SignalingTechnology); anti-Flag (clone F3165), anti-actin (clone A2066), anti-tubulin (clone T6199) (Sigma-Aldrich); anti-PI3K3R1 (clone 06-195) (Upstate); and anti-IRS2 (Millipore MABS15). The phosphospecific antibody against IRS2 Ser388 was synthesized and purchased from GenScript.
Activin and Notch signaling are both indispensable for proper early development. Activin-like signaling plays important roles in the induction of dorsal mesoderm during early embryogenesis. On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. We specu- lated that during activin A induces various tissues from homog- enous undifferentiated cells, Notch signaling is activated and plays some roles. Our results revealed that, in animal caps, activin A induced the expression of Delta-1 , Delta-2 , and Notch , followed by the expression of ESR-1 . The delay of ESR-1 expression compared with Delta-1 , Delta-2 , and Notch suggested that activin A first induced Delta-1 , Delta-2 , and Notch expression, which activated Notch signaling, and then ESR-1 expression was in- duced (Fig. 5). Our real-time RT-PCR analysis showed that there is an optimal concentration of activin A for inducing Delta-1 and Delta-2 . Activin A at intermediate concentrations (1 and 10 ng/ml) was more efficient in inducing Delta-1 and Delta-2 expression than the lower- and higher-range concentrations (0.1 and 100 ng/ ml, respectively). In stage-11 embryos, Delta-1 and Delta-2 are expressed in the marginal zone, in a ring around the blastopore. These findings suggest that the dose-dependent induction may
an existing in vivo measurement technology, FSCV, with a carbon-fiber microelectrode (CFM) as the sensor. With FSCV, the change in concentration of many electroactive molecules can be recorded and displayed. FSCV protocols for measuring DA , , , , ,  – , ,  – , hydrogen peroxide , , , oxygen , , norepinephrine , , serotonin ,  and pH ,  have all be reported. In FSCV, a potential waveform is applied to a working electrode, generating a current response. The recorded currents are typically displayed versus the applied potential as a cyclic voltammogram (CV). CVs offer valuable insight into the redox reactions that occur at the surface of the electrode by creating a chemical fingerprint of the molecule. This fingerprint contains both the identity and concentration of the species derived from the peak location and magnitude, respectively . FSCV has been utilized in vivo for many years. As this technology moves toward longer-term experiments and use in freely moving animals, there is a need to improve the engineering performance of FSCV. Existing FSCV protocols were developed initially for use in narrowly defined, acute experimentation, thus little consideration was placed on data transfer requirements. In a typical experiment the electrochemical information is oversampled leading to large volumes of data. Limitations in data transfer hinders the ability to move towards a system that takes measurements over a longer period of time as well as the application of a wireless system that would provide animals greater freedom of motion.
ty pathway and the Wnt/Ca2+ pathway . The canonical Wnt signaling pathway also named the Wnt/β-catenin pathway was consid- ered as the most prevalent mechanism in the development of cancer, and its activation was a highly integrated process with complex multiple steps involving in Wnt2, CK1, Axin, APC, β-catenin, TCF, TAK1 and SMAD4. Wnt2, as a member of DEPs, binded with the Fzd receptor on the membrane to initiate the canonical Wnt signaling pathway. CK1, Axin, APC and GSK-3β formed a complex of Axin, priming β-catenin for further phosphorylation by GSK3β and subse- quent degradation . Stabilized β-catenin translocated from membrane to nucleus and activated the expression of TCF/LEF (T cell fac- tor/lymphoid enhancing factor)-triggered target genes . TAK1 constituted a part of pro- inflammatory, activating NF-kappa B and MAPK pathways . However, the study of them in ESCC was little reported.
Methods: We used a systems biology approach to identify potential key regulatory factors in smoking-induced lung cancer. We first identified genes that were differentially expressed between smokers with normal lungs and those with cancerous lungs, then integrated these differentially expressed genes (DEGs) with data from a protein-protein interaction database to build a network model with functional modules for pathway analysis. We also carried out a gene set enrichment analysis of DEG lists using the Kinase Enrichment Analysis (KEA), Protein-Protein Interaction (PPI) hubs, and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. Results: Twelve transcription factors were identified as having potential significance in lung cancer (CREB1, NUCKS1, HOXB4, MYCN, MYC, PHF8, TRIM28, WT1, CUX1, CRX, GABP, and TCF3); three of these (CRX, GABP, and TCF) have not been previously implicated in lung carcinogenesis. In addition, 11 kinases were found to be potentially related to lung cancer (MAPK1, IGF1R, RPS6KA1, ATR, MAPK14, MAPK3, MAPK4, MAPK8, PRKCZ, and INSR, and PRKAA1). However, PRKAA1 is reported here for the first time. MEPCE, CDK1, PRKCA, COPS5, GSK3B, BRCA1, EP300, and PIN1 were identified as potential hubs in lung cancer-associated signaling. In addition, we found 18 pathways that were potentially related to lung carcinogenesis, of which 12 (mitogen-activated protein kinase, gonadotropin-releasing hormone, Toll-like receptor, ErbB, and insulin signaling; purine and ether lipid metabolism; adherens junctions; regulation of autophagy; snare interactions in vesicular transport; and cell cycle) have been previously identified.
Moreover, as shown in Figure 2, the GC meta- signature miRNAs target genes were enriched in cell signaling pathways, such as HIF-1, FoxO, sphingolipid and PI3K-Akt Aberrations in such signaling pathways and their contribution to malignancy development are discussed thoroughly in the literature (46-49). From molecular function enrichment analysis, it is indicates that these meta- signature microRNAs regulate cancer cell behavior. Through modulating cell mobility, cell fate determination and cancer cell metabolism modulation, development of malignancy phenotype would be tuned.
In mammals, sonic hedgehog (Shh) is expressed in the notochord and floor plate of the developing spinal cord, establishing a dorsoventral gradient of Hh activity that is required for ventral neural cell fates including motoneurons (Ericson et al., 1995; Roelink et al., 1995; Chiang et al., 1996). The role of Hh signaling in teleost motoneuron development, however, is less clear. Zebrafish have primary and secondary motoneurons (PMNs and SMNs), which differ in their time of appearance during development (Kimmel and Westerfield, 1990). Elevated Hh signaling induces extra PMNs and SMNs (Chandrasekhar et al., 1998), and Hh signaling is necessary for SMN specification (Varga et al., 2001). Although zebrafish lacking zygotic Smo retain significant numbers of PMNs (Chen et al., 2001; Varga et al., 2001), it has been reported that nearly all PMNs can be eliminated in embryos treated with the Smo antagonist cyclopamine (Chen et al., 2001) or injected with morpholinos (MOs) targeting Hh ligands (Lewis and Eisen, 2001). These observations have led to the hypothesis that PMNs remaining in zygotic smo mutants are specified by maternal smo activity (Chen et al., 2001; Varga et al., 2001).
We are used to thinking of signaling as being in-band. We hear dial tone, dial digits, and hear ringing over the same channel on the same pair of wires. When the call completes, we talk over the same path that was used for the signaling. Traditional telephony used to work in this way as well. The signals to set up a call between one switch and another always took place over the same trunk that would eventually carry the call. Signaling took the form of a series of multifrequency (MF) tones, much like touch tone dialing between switches.
pathway, Neurotrophin signaling pathway, MAPK signaling pathway and so on. PPARs act as nuclear receptor and its activation induces a decrease in neuronal death by prevention of oxidative or inflammatory mechanisms implicated in cerebral in- jury . Neurotrophins are a family of trophic fac- tors involved in differentiation and survival of neural cells, which have now been shown to mediate both positive and negative survival signals, by signalling through the Trk and p75 neurotrophin receptors, re- spectively [34, 35]. TGF-βs regulate a wide spectrum of cellular functions such as proliferation, apoptosis, differentiation and migration. TGF-beta signaling is a molecular mechanism which limit neuroinflammation, and activate TGF-beta in the peri-infarct cortex and preserve brain function during the subacute period after stroke [11, 36]. The MAP-kinase family mem- bers are known to be stimulated after cerebral ische- mia and were thought to regulate signal transduction, gene expression and metabolism. Based on the above information, we hypothesize that these differentially expressed miRNAs serve as mediators of the brain’s response to FNS that leads to endogenous neuropro- tection. On the other hand, our research investigated the role of miR-29c-3p in the neuroprotection in- duced by FNS after ischemic injury and we found that miR-29c-3p attenuated ischemic neuronal death by negatively regulating apoptotic proteins Birc2 and Bak1 associated with the PI3K-Akt signaling pathway . Our finding expand the understanding of miR- NAs associated with ischemic cerebral disease and may provide a basis for novel therapeutic strategies aimed at enhancing tissue and cell survival in the is- chemic stroke.
and -deficient populations . Although variations permeate the literature, vitamin D status is generally considered to be deficient when 25(OH)D serum concentrations are <25 nmol/L, insufficient when between 25 and 49 nmol/L, and sufficient when ≥ 50 nmol/L . Vitamin D deficiency is linked to the development of several pathologies including cancer, autoimmune diseases, infectious diseases, and inflammatory conditions . In addition, previous reports suggest that vitamin D plays a role in glycemic control [8,9] which can be improved through vitamin D supplementation [8–10]; however, findings are contradictory [11–13]. Potential cellular pathways are unclear, but may occur through direct binding of vitamin D to vitamin D receptors and activation of downstream signaling proteins; through increased gene expression of the insulin receptor; and indirectly through calcium regulation and subsequent downstream effects on glucose homeostasis signaling proteins [14–17]. Previous reports indicate that vitamin D supplementation in high-fat diet–fed mice attenuates weight gain and increases transcriptional activity of the insulin receptor substrate-1 (IRS-1) in skeletal muscle but not adipose tissue . Likewise, 1,25(OH) 2 D treatment in C2C12 cells rescues diet-induced
carboxamide ribonucleoside-induced activation of AMPK in knockin mice expressing a glucose-6-phosphate-insensitive glycogen synthase mutant demonstrates elevated glycogen accumulation through allosteric activation of glycogen syn- thase, caused by elevated glucose uptake and intracellular glucose-6-phosphate levels. This evidence suggests that AMPK may be able to promote glycogen synthesis in skel- etal muscle independent of the traditional insulin-signaling nexus. 42 More work is clearly needed to identify how, or even
Many studies on the TOR signaling pathway have provided invaluable insights into the regulation and function of this pathway. While TOR signaling is an extensively studied pathway, the majority of studies done were on the TORC1 signaling branch. Much remains unknown about the regulation and possible novel functions of the TORC2 signaling branch. Besides Tor2, Lst8 is the only other essential component of both TOR signaling complexes. I have addressed two questions. First, the function of Lst8 in these two complexes is unknown and whether the essential function of Lst8 is through its action in TORC1 or TORC2 or both remains a mystery. Solving this mystery could provide insights into the essential function of Lst8 in TOR signaling. It is not possible to study the effect of losing an essential gene because cells will die in the absence of that gene product. Therefore, I addressed the following question: Are there mutations that will allow cells to survive without Lst8? Identification of such mutants would allow further studies to be performed on the mutants to address the first
The ecdysone pulse has been shown to act non-autono- mously to affect larval growth. These cell extrinsic effects of the ecdysone pathway are reviewed elsewhere [38-41] and therefore only mentioned briefly here. This control of Drosophila larval growth and final body size occurs non- autonomously, at least in part through interactions between the ecdysone and insulin pathways. The insulin signaling pathway acts in the prothoracic gland (PG) to regulate the release of ecdysone, therefore influencing the rate and duration of larval growth [38,39,42-45]. For instance, increased PG growth occurs when PI3-kinase (PI3K, a downstream regulator of the insulin pathway) is upregulated in the PG [42,44]. The PG overgrowth causes accelerated metamorphosis, which results in reduced adult size due to the rapid progression through the larval growth stage. Precocious ecdysone release, as measured by premature increase in levels of the early response ecdys- one genes, correlates with this disruption to larval growth. Conversely, reducing growth of the PG, using a dominant negative form of PI3K, resulted in a longer larval growth period and larger adults due to slower ecdysone release and delayed onset of pupariation. More recently it has been shown that Target of Rapamycin (TOR) may link the ecdysone regulated development to the PI3K mediated growth pathways  (reviewed ).
Certain autoimmune diseases, such RA and SLE, arise from an inappropriate immune response of the body against self-antigens [1,30,31]. SLE, for instance, is charac- terized by the loss of tolerance to self-nuclear antigens, the deposition of immune complexes in tissues, and multiorgan involvement . Studies have shown that nuclear-acid sensing pathways implicated in the subversion of the innate immune response to discriminate between self-antigen and foreign antigens are those mediated by the TLRs in the context of SLE pathogenesis [32-34]. BTK, which is a downstream kinase of SYK, has been implicated in TLR signaling recently [24,25], whereas the role of SYK in TLR signaling is not well appreciated. It has been reported that the TLR9 agonist CpG could induce TLR-9 independent SYK phosphorylation and activation through actin cytoskel- eton reorganization, leading to activation of Src family kinases . Recruitment of SYK to TLR9 and phosphoryl- ation of TLR9 are required for CpG-induced cytokine pro- duction. We therefore used RO9021 to study the role of SYK in TLR9 signaling. Interestingly, the kinase function of SYK is essential for TLR9-mediated responses in human B cells (Figure 5A,B,C). Inhibition of SYK kinase function resulted in a decreased level of plasmablasts, IgM, IgG and IL-6 upon B-cell differentiation in the presence of TLR9 ligand. In addition, RO9021 also potently inhibited IFNα production by human pDCs upon TLR9 activation (Figure 5D). Importantly, the effects on TLR9 responses are specific because RO9021 did not inhibit TLR4-dependent TNFα production by human monocytes (Figure 2E) or acti- vation of the JAK–STAT pathway stimulated with either IL-2 or IFNγ (Figure 3). Our study showed for the first time that kinase activity of SYK is critical for TLR9 signaling pathway in B cells and pDCs.