Journal: bioRxiv

Article Title: Casein Kinase II is Required for Proper Cell Division and Acts as a Negative Regulator of Centrosome Duplication in C. elegans Embryos

doi: 10.1101/083378

Figure Lengend Snippet: (A) TBB treatment restores embryonic viability to zyg-1(it25) animals at 22.5°C. (DMSO control: 3 ± 3%; TBB: 37 ± 22%), but TBB had a mild effect on wild-type worms (DMSO: 95 ± 6%; TBB: 92 ± 8%). (B) TBB treatment leads to a significant reduction in brood size [wild-type (DMSO: 117 ± 28; TBB: 49 ± 11), zyg-1(it25) (DMSO: 67 ± 23; TBB: 39 ± 13)] (A, B) Each dot represents an animal. (C) TBB-treated zyg-1(it25) embryo exhibits bipolar spindles, but DMSO control embryo with monopolar spindles. (D) TBB treatment enhances bipolar spindle formation in zyg-1(it25) embryos (DMSO: 32 ± 16%; TBB: 73 ± 9.5%, n=blastomeres). Error bars are s.d. (E) Quantification of centrosomal ZYG-1 levels in TBB-treated wild-type embryos. Each dot represents a centrosome. (A,B,E) Box ranges from the first through third quartile of the data. Thick bar represents the median. Dashed line extends 1.5 times the inter-quartile range or to the minimum and maximum data point. (F) TBB-treated wild-type embryo shows more intense ZYG-1 focus at centrosomes. Insets illustrate centrosomal regions magnified 4 folds. (G) Wild-type embryos treated with TBB exhibit defective cell divisions that are similar to RNAi-mediated depletion of CK2: (a) lagging DNA (boxes: Insets are 2 folds), (b) expanded PCM (boxes), (c) detached centrosomes (arrow), (d) cell cycle delay: note cell cycle stages in AB (left) relative to P1 (right) cell. Shown are still images from live confocal imaging. *** p <0.001, ** p <0.01 (two-tailed t-test). Scale bar, 10 μm.

Article Snippet: The media was then supplemented with 0.5 mM TBB (Tocris, Avonmouth, Bristol, UK) dissolved in a solution of 50% DMSO.

Techniques: Control, Imaging, Two Tailed Test

Journal: PLoS ONE

Article Title: Site-specific HNK-1 epitope on alternatively spliced fibronectin type-III repeats in tenascin-C promotes neurite outgrowth of hippocampal neurons through contactin-1

doi: 10.1371/journal.pone.0210193

Figure Lengend Snippet: Primary hippocampal neurons were cultured on PDL, PDL coated with FNx7 without HNK-1 expression (HNK-1(-)), or PDL coated with FNx7 with HNK-1 expression (HNK-1(+)). Normal goat IgG (+ normal IgG) or anti-CNTN pAb (+ CNTN Ab) was added to the culture medium at a final concentration of 30 μg/mL after 18 h of culture. Neurons were immunostained and analyzed at DIV3. Green, PDL with normal goat IgG (n = 219); light green, PDL with anti-CNTN pAb (n = 130); yellow, FNx7 HNK-1(-) with normal goat IgG (n = 139); light yellow, FNx7 HNK-1(-) with anti-CNTN pAb (n = 163); orange, FNx7 HNK-1(+) with normal goat IgG (n = 154); and light orange, FNx7 HNK-1(+) with anti-CNTN pAb (n = 166). Numbers of measured neurons were indicated in parentheses. This experiment was performed across two independent cultures, respectively, and the representative result obtained from a single culture was shown. Error bars represent SEM. ***p < 0.001; n.s., p > 0.05.

Article Snippet: At day 3 in vitro (DIV3), hippocampal primary neurons were fixed in methanol at -20°C for 20 min and incubated with mouse anti-Tau-1 monoclonal antibody (mAb) (5 μg/mL, MAB3420 [AB_94855]; Millipore, Stafford, VA, USA) in PBS containing 3% BSA for 1 h at room temperature, followed by incubation with Alexa Fluor 546-conjugated donkey anti-mouse IgG antibody (5 μg/mL, A10036 [AB_2534012]; Molecular Probes, Eugene, OR, USA) in PBS containing 3% BSA at room temperature for 1 h. For the application of antibodies in culture medium, 30 μg/mL control goat IgG (sc-2028 [AB_737167]; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-contactin-1 polyclonal antibody (pAb) were added after 18 h of culture.

Techniques: Cell Culture, Expressing, Concentration Assay