Journal: Frontiers in Molecular Neuroscience

Article Title: Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons

doi: 10.3389/fnmol.2024.1393779

Figure Lengend Snippet: Ptbp2 interacts with Smn in motoneurons. (A) Representative images of Smn-Ptbp2 PLA signal in cultured motoneurons at DIV 6 using anti-Smn and anti-Ptbp2 antibodies. Motoneuron morphology was visualized with anti-Tubb3 antibody. Scale bars, 10 and 5 μm (magnified areas). (B) Co-immunoprecipitation of Smn by anti-Ptbp2 from motoneuron lysate pre-treated with RNase A as indicated. (C) Representative images showing Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Arrowheads indicate colocalization of Smn and Hnrnpr in granules. Scale bars, 10 and 2 μm (magnified areas). (D) Fluorescence intensity profiles of Smn and Hnrnpr at the location indicated by arrow 4 in (C) . (E) Representative images showing Ptbp2 and Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Scale bars, 10 and 2 μm (magnified areas). (F) Fluorescence intensity profiles of Ptbp2, Smn and Hnrnpr at the location indicated by a line in (E) .

Article Snippet: Cells were fixed again for 10 min at room temperature in PLP, washed with DPBS, and stained with FITC-conjugated anti-Tubb3 antibody (130-131-158, Miltenyi Biotec).

Techniques: Cell Culture, Immunoprecipitation, Immunofluorescence, Fluorescence

Journal: Communications Biology

Article Title: Precise CAG repeat contraction in a Huntington’s Disease mouse model is enabled by gene editing with SpCas9-NG

doi: 10.1038/s42003-021-02304-w

Figure Lengend Snippet: a The procedure of neuronal differentiation of ES cells. For in vitro differentiation assay, we used ES clones transfected with prototype SpCas9-NG (not published), not ES clones described in Fig. . b Immunostaining of differentiated embryoid bodies. White arrows in the high magnified image of R6/2 clone #2 indicate HTT aggregates. All scale bars indicate 100 µm: the EB in genome-edited #2 (top) was almost twice larger than the others. c The differentiation scores based on a percentage of the β III tubulin staining in the circumference of embryoid bodies; 1: 0–25%; 2: 25–50%; 3: 50–75%; 4: 75–100%. Statistical analysis was performed using a two-tailed Mann–Whitney U-test ( p -value = 0.012).

Article Snippet: After three washes with PBS, the cells were incubated with Alexa Fluor 555 conjugated goat anti-mouse IgG (A21422, Thermo Fisher Scientific; 1:4000) at room temperature for 2 h. The cells were then incubated with a mouse anti-β III tubulin mouse antibody (T8660, Merck; 1:1000) at 4 °C overnight.

Techniques: In Vitro, Differentiation Assay, Clone Assay, Transfection, Immunostaining, Staining, Two Tailed Test, MANN-WHITNEY

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

Journal: Frontiers in Cellular Neuroscience

Article Title: Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses

doi: 10.3389/fncel.2014.00105

Figure Lengend Snippet: Quantification of DRG explant neurite outgrowth in response to culture media . M+GF (DMEM+ growth factors), M-GF (DMEM without growth factors), and conditioned media: CM CTR (control spinal cord), after SCI: CM RSCI (rostral segment), CM LSCI (lesioned segment), CM CSC (caudal segment) (A) . The image of TUJ1 stained DRG explants after selecting the area to be analyzed and thresholding the number of pixels covered by the extending neuritis, but not the DRG cell body (asterisk) (B) . Representative immunofluorescence images of TUJ1 positive DRG explants exposed to CM RSCI (C) , CM LSCI (D) , and CM CSC (E) . Scale bars (B–E) = 400 μm.

Article Snippet: Briefly, DRGs were incubated in mouse anti-Neuron-specific class III beta-tubulin (TUJ1) (1:200; Merck) for 24 h at 4°C.

Techniques: Staining, Immunofluorescence