The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer 1-5. Inflammasomes are multiprotein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to

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• The ABC transporter, ABCC4, localizes to the platelet plasma membrane and regulates aggregation by exporting cAMP and antithrombotic drugs.Controlling the activation of platelets is a key strategy to mitigate cardiovascular disease. Previous studies have suggested that the ATP-binding cassette (ABC) transporter, ABCC4, functions in platelet-dense granules. Using plasma membrane biotinylation and superresolution microscopy, we demonstrate that ABCC4 is primarily expressed on the plasma membrane of both mouse and human platelets. Platelets lacking ABCC4 have unchanged dense-granule function, number, and volume, but harbor a selective impairment in collagen-induced aggregation. Accordingly, Abcc4 knockout (KO) platelet attachment to a collagen substratum was also faulty and associated with elevated intracellular cyclic AMP (cAMP) and reduced plasma membrane localization of the major collagen receptor, GPVI. In the ferric-chloride vasculature injury model, Abcc4 KO mice exhibited markedly impaired thrombus formation. The attenuation of platelet aggregation by the phosphodiesterase inhibitor EHNA (a non-ABCC4 substrate), when combined with Abcc4 deficiency, illustrated a crucial functional interaction between phosphodiesterases and ABCC4. This was extended in vivo where EHNA dramatically prolonged the bleeding time, but only in Abcc4 KO mice. Further, we demonstrated in human platelets that ABCC4 inhibition, when coupled with phosphodiesterase inhibition, strongly impaired platelet aggregation. These findings have important clinical implications because they directly highlight an important relationship between ABCC4 transporter function and phosphodiesterases in accounting for the cAMPdirected activity of antithrombotic agents. (Blood. 2015;126(20):2307-2319

We have shown previously that, in astrocytoma cells, synemin is present at the leading edge, an unusual localization for an intermediate filament (IF) protein. Here, we report that synemin down-regulation with specific small hairpin RNAs (shRNAs) sharply decreased the migration of astrocytoma cells. The presence of synemin at the leading edge also correlated with a high migratory potential, as shown by comparing astrocytoma cells to carcinoma cells without synemin at the leading edge. Synemin-silenced astrocytoma cells were smaller and spread more slowly than controls. In addition, synemin silencing reduced proliferation without increasing apoptosis. The adhesion to substratum and distribution of vinculin in focal contacts of synemin-silenced astrocytoma cells were similar to those of controls. Synemin-silenced cells, however, exhibited a reduction in the amount of filamentous (F) -actin and of alpha-actinin, but not of vinculin, associated with F-actin. Altogether, these results demonstrate that synemin is important for the malignant behavior of astrocytoma cells and that it contributes to the high motility of these cells by modulating the dynamics of alpha-actinin and actin.

Synemin is an intermediate filament protein present in glioblastomas (GBMs) but not in normal brain. In GBM cells synemin interacts with and antagonizes PP2A, which is the phosphatase dephosphorylating Akt. This maintains the phosphorylation status of Akt sites that are substrates for PDPK1 and mTORc2, thereby fostering proliferation.

Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse neonatal survival and is estimated to impact between 0.4 and 5% of pregnancies worldwide. Here we show that maternal cholestasis (due to Abcb11 deficiency) produces neonatal death among all offspring within 24 h of birth due to atelectasis-producing pulmonary hypoxia, which recapitulates the neonatal respiratory distress of human ICP. Neonates of Abcb11-deficient mothers have elevated pulmonary bile acids and altered pulmonary surfactant structure. Maternal absence of Nr1i2 superimposed on Abcb11 deficiency strongly reduces maternal serum bile acid concentrations and increases neonatal survival. We identify pulmonary bile acids as a key factor in the disruption of the structure of pulmonary surfactant in neonates of ICP. These findings have important implications for neonatal respiratory failure, especially when maternal bile acids are elevated during pregnancy, and highlight potential pathways and targets amenable to therapeutic intervention to ameliorate this condition.

Relative changes in the expression of a specific protein are commonly measured on western blots by forming the ratio of the densitometric values of bands containing the protein between control and experimental samples. It is generally assumed that this analysis provides an accurate determination of relative changes in a specific protein expression if there is a linear relation between increasing amounts of that protein, as represented by bands on a western blot or a gel, and the densitometric measurements of these bands. In this study, we provide direct evidence that this assumption is invalid because even in the presence of a linear relation, densitometric ratios differ substantially from know actual ratios of protein amounts. We present also the mathematical basis for this bias, and demonstrate that it can be circumvented by using a "ratio standard curve". Alternatively, the use of purified protein standards to plot a standard densitometry curve can solve this problem, but a survey of the literature shows that most investigators who use western blots to quantify relative changes in protein expression samples do not have standards for the specific proteins that they are investigating.To examine whether densitometric ratios accurately reflect ratios between protein amounts, we performed experiments to silence lamin C (a nuclear envelope protein) in U-373 MG glioblastoma cells. These cells were grown as described earlier [1], and were transfected with a lamin C specific siRNA (5′-CUGGACUUCCAGAAGAdTdT-3′ and 5′-UGUUCUUCUGGAAGUCCAGdTdT-3′ for the sense and anti-sense sequences, respectively) using siLentFect lipid reagent (Bio-Rad) according to the manufacturer's instructions. Control cells were treated with transfection reagent and vehicle. One and two days after transfection, the cells were processed for SDS-PAGE as described earlier [1]. Protein concentrations were measured with the bicinchoninic acid assay [2]. Gels were loaded with incremental amounts (4, 8, 20, 40, and 80 g) of total cellular proteins from control samples, as well as with 80 g of total cellular protein for experimental samples. Each loading was performed in duplicate, and all the loadings were applied to the same gel to insure identical treatment during the western blotting procedure, which was performed following standard methods [3]. In addition, to avoid band distortion, sample loadings were brought to the same final volume with sample buffer. After blocking in PBS plus 5% dry milk, the blots were incubated with a rabbit anti-lamin antibody (Chemicon, Temecula, CA) diluted at 1:100 in blocking solution, and then with goat anti-rabbit IgGs conjugated to peroxidase (KPL, Gaithersburg, MD) diluted at 1:1000 in blocking solution. After incubation of the blots Address correspondance to: Dr.

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