Journal: Molecular neurobiology

Article Title: Microtubule-Actin Crosslinking Factor 1 is required for dendritic arborization and axon outgrowth in the developing brain

doi: 10.1007/s12035-015-9508-4

Figure Lengend Snippet: (A) MACF1 expression in cortical neurons. Cortical neurons from E14.5 control (MACF1loxP/+; Nex-cre) and MACF1loxP/loxP; Nex-cre brains were cultured for 5 days, and MACF1 localization was examined using immunostaining. Arrows indicate an accumulation of MACF1 in dendritic tips (B) MACF1 deletion causes aberrant actin arrangement and localization. Polymerized actins were visualized by phalloidin staining. Top panels show the patterns of polymerized actins at neurite tips. Middel and bottom panels show polymerized actins accumulated in the cytosol. (C) The levels of polymerized actin (F-actin) intensity in the cell body and at the neurite tip were quantified using Image J (NIH) software. n = 21 cells from 3 independent cultures using 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001. (D) MACF1-deleted neurons show abnormal microtubule arrangement at the neurite tip. (E) Immunoblotting was performed to measure the levels of α-tubulin or acetylated-tubulin using E14.5 control and MACF1loxP/loxP; Nex-cre brain lysates (top panel). The levels of each tubulin were quantified in the bottom panel. n = 3 blots using 3 different lysates from 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001.

Article Snippet: The following primary antibodies were used: Chicken anti-GFP (Invitrogen), Rabbit anti-GFP (Invitrogen), mouse anti-β-III Tubulin (Phosphosolutions), rabbit anti-acetyl-α-tubulin (Cell Signaling), and mouse anti-β-tubulin (Upstate).

Techniques: Expressing, Control, Cell Culture, Immunostaining, Staining, Software, Two Tailed Test, Western Blot

Journal: PLoS ONE

Article Title: No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

doi: 10.1371/journal.pone.0118947

Figure Lengend Snippet: A-B: Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; C-H: Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. C1-H2: Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.

Article Snippet: At day in vitro (DIV) 3, 7 and 14 neuronal cultures were immunostained with mouse anti-ß-Tubulin, Class III antibody conjugated to Alexa Fluor 488 (1:50; BD Pharmingen) and the nuclear marker Hoechst 33342 (1:2000), both diluted in PBS.

Techniques: Transgenic Assay, Staining, Derivative Assay, Software