Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: PLOS Pathogens

Article Title: PIP 2 regulating calcium signal modulates actin cytoskeleton-dependent cytoadherence and cytolytic capacity in the protozoan parasite Trichomonas vaginalis

doi: 10.1371/journal.ppat.1011891

Figure Lengend Snippet: The TH17 trophozoites without (Control) or with HA- Tv PI4P5K Wt or K136A overexpression pretreated with DMSO, Edelfosine, BAPTA-AM, or LatB were inoculated into h VECs in the presence or absence of a protease inhibitor cocktail (PIC). (A) The parasites were co-cultured with h VECs at different MOI for 1 hr in the LDH cytotoxicity assay. The assay was performed in three biological repeats to measure the relative cytotoxicity (%) as shown in the bar graph (n = 3, mean ± SD). (B) The trophozoites labeled with CMRA were co-cultured with h VECs (MOI = 4) for 1 hr. All specimens were fixed for staining with FITC-conjugated phalloidin. Nuclei were stained with DAPI. The white dashed line indicates the area lysed by the parasite on the h VECs monolayer. The assay was performed in three biological repeats, and the average lysis area per trophozoite is shown in the Box-whisker plot (n = 5, mean ± SD). The significant differences of grouped samples were analyzed by Student’s t-tests with the p< 0.05(*), p< 0.01(**), and ns, no significant difference.

Article Snippet: The supernatant was assayed using the LDH cytotoxicity assay kit (Biochain).

Techniques: Over Expression, Protease Inhibitor, Cell Culture, LDH Cytotoxicity Assay, Labeling, Staining, Lysis, Whisker Assay