Journal: PLoS ONE

Article Title: Modeling Chemotherapeutic Neurotoxicity with Human Induced Pluripotent Stem Cell-Derived Neuronal Cells

doi: 10.1371/journal.pone.0118020

Figure Lengend Snippet: Relative viability measured by CellTiter-Glo (black lines, right Y-axes) was significantly higher than relative total outgrowth (colored lines, left Y-axes) upon treatment of iCell Neurons for 72 h with (A) paclitaxel (Welch's t-test of AUC P = 0.0005) and (B) vincristine (P = 0.027), but not (C) cisplatin (P = 0.71). Three replicates of the total outgrowth assay and four replicates of the viability assay were performed. (D) Relative caspase 3/7 activity measured by Caspase-Glo 3/7 after 48 h treatment significantly differed among drugs (one-way ANOVA of AUC calculated from 0.001–10 μM P = 0.002). Five replicates of the apoptosis assay were performed. (E) Paclitaxel-induced decrease in neurite outgrowth of iCell Neurons is influenced by TUBB2A expression. Left, relative TUBB2A expression 24 to 48 h post-transfection. Right, decreased expression of TUBB2A by siRNA transfection causes a greater decrease in relative total neurite outgrowth of iCell Neurons (Welch's t-test P = 0.011) 24 h post-treatment with 0.1 μM paclitaxel (48 h post-transfection). Error bars represent the standard error of the mean from three independent transfection experiments. NTC = non-targeting control.

Article Snippet: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the level of expression of TUBB2A (Hs00742533_s1) 24 and 48 h after transfection.

Techniques: Viability Assay, Activity Assay, Apoptosis Assay, Expressing, Transfection, Control

Journal: Neuroscience

Article Title: Altered cytoskeletal composition and delayed neurite elongation in tau 45-230 -expressing hippocampal neurons

doi: 10.1016/j.neuroscience.2019.05.046

Figure Lengend Snippet: Quantitative Western blot analysis of whole hippocampus homogenates [Triton X-100 (−)] and cytoskeletal fraction samples [Triton X-100 (+)] obtained from 3-, 6-, and 9-month postnatal wild type (WT) and tau45-230-transgenic (T) mice were reacted with tau (clone tau5) (A), dephosphorylated tau (clone tau1) (B), and tubulin antibodies (C & D). Samples were normalized to α-tubulin as protein controls. Values represent the mean ± S.E.M. from 5 independent experiments per condition. The levels of a given protein in WT controls were considered 100%.

Article Snippet: Lysates were loaded equally based on internal control levels of α-tubulin (for whole cell extracts) or acetylated tubulin (for cytoskeleton fractions).

Techniques: Western Blot, Transgenic Assay

Journal: Neuroscience

Article Title: Altered cytoskeletal composition and delayed neurite elongation in tau 45-230 -expressing hippocampal neurons

doi: 10.1016/j.neuroscience.2019.05.046

Figure Lengend Snippet: Quantitative Western blot analysis of cytoskeletal fraction samples prepared from wild type (WT) and tau45-230 transgenic (T) cultured hippocampal neurons at 1, 7, and 14 days after plating were reacted with tau (clone tau 5) (A), dephosphorylated tau (clone tau1) (B), and tubulin antibodies (C & D). Cytoskeletal fraction samples were normalized to acetylated tubulin as protein controls. Values represent the mean ± S.E.M. from 3 independent experiments per condition. The levels of a given protein in WT controls were considered 100%. *Differs from WT control values, p < 0.05.

Article Snippet: Lysates were loaded equally based on internal control levels of α-tubulin (for whole cell extracts) or acetylated tubulin (for cytoskeleton fractions).

Techniques: Western Blot, Transgenic Assay, Cell Culture

Journal: Neuroscience

Article Title: Altered cytoskeletal composition and delayed neurite elongation in tau 45-230 -expressing hippocampal neurons

doi: 10.1016/j.neuroscience.2019.05.046

Figure Lengend Snippet: (A-F) Quantitative Western blot analysis of cytoskeletal fraction samples obtained from 2 days in culture control and tau45-230-transfected hippocampal neurons reacted with GFP (A), tau (clone tau5) (B), dephosphorylated tau (clone tau1) (C), and tubulin antibodies (D-F). Cytoskeletal fraction samples were normalized to acetylated tubulin as protein controls. *Indicates endogenous full-length tau immunoreactive band. Values represent the mean ± S.E.M. from 10 independent experiments per condition. The levels of a given protein in non-transfected controls were considered 100%. *Differs from control values p < 0.05.

Article Snippet: Lysates were loaded equally based on internal control levels of α-tubulin (for whole cell extracts) or acetylated tubulin (for cytoskeleton fractions).

Techniques: Western Blot, Transfection

Journal: Neuroscience

Article Title: Altered cytoskeletal composition and delayed neurite elongation in tau 45-230 -expressing hippocampal neurons

doi: 10.1016/j.neuroscience.2019.05.046

Figure Lengend Snippet: (A & B) Tau45-230-transfected cultures were fixed and double-stained using GFP (A) and tubulin (B) antibodies. Most of GFP-tau45-230 (+) neurons (arrows) remained in stage 1 and 2, failing to elongate their axons after 2 days in culture. Scale bar: 20 μm. (C) Quantification of tau45-230 (−) and tau45-230 (+) neurons in stage 1, 2, and 3. Neurons in 30 fields from 3 independent culture preparations per experimental condition were counted. **Differs from untransfected controls, p < 0.01. ax: axon

Article Snippet: Lysates were loaded equally based on internal control levels of α-tubulin (for whole cell extracts) or acetylated tubulin (for cytoskeleton fractions).

Techniques: Transfection, Staining

Journal: Molecular Biology of the Cell

Article Title: Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

doi: 10.1091/mbc.E16-08-0571

Figure Lengend Snippet: Pigment granule aggregation signals increase nucleation of MTs on pigment granules, and this increase correlates with the recruitment of γ-tubulin. (A) Fluorescence (left) and phase contrast (right) images of pellets of immunostained MTs assembled by polymerization of twice-cycled tubulin incubated alone or with different pigment granule preparations; shown are images from (Tb) alone, (PG(D)+Tb) pigment granules isolated from cells treated with MSH to induce granule dispersion, (PG(A)+Tb) pigment granules from cells treated with melatonin to trigger aggregation and preincubated with buffer only, (PG(A)+IgG+Tb) pretreated with nonimmune rabbit IgG, (PG(A)+ γ-Tb antibody+Tb) pretreated with γ-tubulin antibodies, (PG(A)+CLASP antibody+ Tb) pretreated with CLASP antibodies, or (PG(A)+gatastatin+Tb) pretreated with γ-tubulin inhibitor gatastatin. Pigment granules increased the amount of assembled MTs, and this effect was greater in the case of granules isolated from melatonin- compared with MSH-treated cells; preincubation of granules with γ-tubulin antibodies or gatastatin but not with nonimmune IgG or CLASP antibodies reduced the amount of assembled MTs; bar, 10 μm. (B) Quantification of the number of assembled MTs in samples shown in A; each bar represents the average value of 30 measurements in two independent experiments; the data are expressed as percentage of average number of MTs assembled in the presence of pigment granules isolated from melatonin-treated cells and incubated with relevant buffer, which is taken as 100%; error bars are mean ± SEM. (C) Comparison of MT nucleation activity of pigment granules isolated from melanophores treated with MSH or melatonin by immunoblotting of granule pellets with α-tubulin antibodies; blots with α-tubulin (top) or TYRP1 (bottom; loading control) antibodies of pellets from samples containing pigment granules isolated from melatonin-treated cells (PG(A)), purified tubulin (Tb), or mixtures of purified tubulin with pigment granules isolated from MSH- or melatonin-treated melanophores (PG(D)+Tb, and PG(A)+Tb, respectively); α-tubulin bands are absent from pellets of pigment granules or tubulin preparations (an indication that pigment granule preparations do not contain significant amounts of tubulin and that MTs do not assemble in the absence of pigment granules) but present in the pellets of mixtures of purified tubulin with pigment granules (an indication of MT nucleation on pigment granules); the amount of α-tubulin is significantly higher in the case of pigment granules isolated from melatonin-treated cells, suggesting stimulation of MT nucleation by the granule-aggregating signals. (D) Immunoblotting of preparations of purified pigment granules isolated from melanophores treated with MSH (left, PG(D)) or melatonin (right, PG(A)) with antibodies against γ-tubulin (left, top), CLASP (right, top) or TYRP1 (loading control; left and right, bottom); γ-tubulin and CLASP levels are increased in preparations of pigment granules isolated from melatonin-treated cells, which suggests that pigment-aggregation signals stimulate recruitment of γ-tubulin and CLASP to pigment granules. (E) Fluorescence images of melanin-free melanophores recovering from MT depolymerization in the absence (left) or presence (right) of gatastatin (30 μM); bottom, high-magnification images of boxed areas shown in the top; gatastatin partially inhibits MT outgrowth from the centrosome; bars, 10 μm (top), 2 μm (bottom). (F) Hypothesis on the stimulation of pigment aggregation by recruitment of γ-tubulin to pigment granules.

Article Snippet: For quantification of MT-nucleation activity of pigment granules by measurement of tubulin levels in granule pellets, a suspension of purified pigment granules was incubated with chromatographically purified porcine brain tubulin (Cytoskeleton) and centrifuged through a solution of 33% glycerol in BRB80 buffer at 1750 × g for 10 min at 37°C.

Techniques: Fluorescence, Incubation, Isolation, Activity Assay, Western Blot, Purification

Journal: Molecular Biology of the Cell

Article Title: Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

doi: 10.1091/mbc.E16-08-0571

Figure Lengend Snippet: Pigment granules nucleate MTs in vivo and in vitro. (A) Sequential live fluorescence images of the centrosome area of an EGFP-EB1–expressing melanophore injected with TYRP1 antibodies and treated with melatonin; an asterisk indicates the position of the centrosome, P shows the location of a clump of pigment granules, and an arrowhead marks an EGFP-EB1 comet that emerges from the pigment granule clump and moves toward the centrosome; numbers indicate time in minutes. Birth of the MT tip labeled with EGFP-EB1 away from the centrosome and its growth toward the cell center suggest nucleation of the MT on a granule clump cross-linked with TYRP1 antibodies; bar, 2 μm. (B) Left and middle, phase contrast (top) and fluorescence (bottom) images of pellets of Cy3-labeled tubulin preparations incubated in the absence (left) or presence (right) of a suspension of purified pigment granules isolated from melanophores treated with melatonin to induce granule aggregation; right, overlays of boxed areas shown in the middle; tubulin pellets that were incubated in the presence of pigment granules contain short MTs that are often attached to pigment granules; bars, 10 μm (left and middle), 2 μm (right).

Article Snippet: For quantification of MT-nucleation activity of pigment granules by measurement of tubulin levels in granule pellets, a suspension of purified pigment granules was incubated with chromatographically purified porcine brain tubulin (Cytoskeleton) and centrifuged through a solution of 33% glycerol in BRB80 buffer at 1750 × g for 10 min at 37°C.

Techniques: In Vivo, In Vitro, Fluorescence, Expressing, Injection, Labeling, Incubation, Purification, Isolation