Journal: bioRxiv

Article Title: Dynamic regulation of NeuroD1 expression level by a novel viral construct during astrocyte-to-neuron reprogramming

doi: 10.1101/2025.02.17.638625

Figure Lengend Snippet: The reprogrammed neurons by the ND1-124T-GFP construct can express markers of mature neurons and neuronal subtypes in long term cultures. ( A ) A live image of HA cell cultures infected by ND1-GFP retrovirus showing floating cell spheroids in the hanging drops of culture medium at 3 DPI. ( B ) Live images of the resulting cell spheroids that are attached to a monolayer of HA cells at 20 DPI. #, the core of the attached spheroid; Arrowhead, a HA cell infected by ND1-GFP that has migrated to periphery of the spheroid showing a typical neuronal morphology. At 30 DPI, the attached HA spheroid cultures infected by either ND1-GFP or ND1-124T-GFP were fixed and immunostained with the indicated mature neuronal markers (NeuN and/or Map2) along with NeuroD1 ( C ), the synaptic vesicle marker SV2 ( D ), the glutamatergic neuronal markers vGlut1 and HuD ( E ), and Ctip2 ( F ). ( G ) Expression levels of the markers were quantified by their cellular fluorescence intensities and compared. ( H ) The GABAergic interneuron markers GABA and GAD67 ( F ) were also analyzed and compared ( I ). Arrows, the marker+ cells. ns, not significant; ***, p < 0.005; ****, p < 0.001 by student t -test. Scale bars, 40μm.

Article Snippet: Briefly, fixed cell cultures were incubated with monoclonal antibodies against NeuroD1 (mouse IgG, 1:1000, Abnova), GFP (rat IgG2a, 1:400, BioLegend), HuD/ELAVL4 (mouse IgG, 1:200, Santa Cruz), GAD67 (mouse IgG, 1:200, Millipore), and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against GFP (chicken IgY, 1:400, Aves), mCherry/RFP (rabbit IgG, 1:500, Abcam), mCherry/RFP (chicken IgY, 1:400, Aves), Map2 (rabbit IgG, 1:400, Abcam), DCX (rabbit IgG, 1:500, Abcam), vGlut1 (guinea pig IgG, 1:200, Millipore), Ctip2 (guinea pig IgG, 1:200, Synapse Systems), anti-SV2 (rabbit, 1:2000, Developmental Studies Hybridoma Bank), GABA (rabbit IgG, 1:200, Calbiochem), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

Techniques: Construct, Infection, Marker, Expressing, Fluorescence

Journal: Cell Reports

Article Title: A cell-type-specific alternative splicing regulator shapes synapse properties in a trans -synaptic manner

doi: 10.1016/j.celrep.2023.112173

Figure Lengend Snippet:

Article Snippet: Additional antibodies are rat anti-HA (Roche, #11867431001, 1:1000), mouse anti-NeuN (Chemicon #MAB377 1:2000), and rabbit anti-CTIP2 (Novus Biologicals, #NB100-2600).

Techniques: Recombinant, Magnetic Beads, Concentration Assay, Reverse Transcription, Western Blot, Fluorescence, Software

Journal: Brain : a journal of neurology

Article Title: NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination and seizure threshold.

doi: 10.1093/brain/awac044

Figure Lengend Snippet: Figure 5 Focal Nprl3 KO in vivo results in lamination defects, increased soma diameter, increased cortical excitability, and reduced seizure threshold. (A and B) GFP+ cells observed in the subcortical white matter (bottom arrow) and in layers IV-VI in Nprl3 KO cortex at P3. In contrast, all GFP+ cells in scramble control specimens (n = 5 per groups; P < 0.001) were observed in the birthdate appropriate layer II/III. GFP+ neurons deep layers in Nprl3 KO specimens did not express CTIP2 indicating that these cells had failed to attain their appropriate laminar destination in layers II/III. GFP+ cells in layer II/III in Nprl3 KO pups were larger than scramble cells in the layer II/III. Rapamycin treatment prevented laminar defects (A and B) and soma diameter increases (C; each box represents mean, SEM, whiskers represent 5–95% confidence intervals). (D) Section from a surgical epilepsy FCD specimen (NeuN-labelled, from Subject 18 in Fig. 1) shows heterotopic white matter neurons (arrows) similar to what was observed in mouse (A and E, arrows). (F) Five weeks after in utero electroporation, mice were implanted with dural EEG electrodes and recorded for 48 h. A representative line length analysis of the 48 h EEG recording shows increased line length (a proxy for spike amplitude) in the electroporated cortex suggesting a hyperexcitable cortex (n = 6 per group) compared with the contralateral cortex and wild-type (WT) mouse cortices, where line lengths in the right and left hemispheres were simi lar. Total average EEG power also showed increased power in Nprl3 KO cortices compared to the contralateral cortex and control cortices (G). Rapamycin treatment after Nprl3 KO rescued increased line length and EEG power (F and G). Mice were treated with 55 mg/kg PTZ and EEGs were recorded. (H) Representative EEG tracings in Nprl3 KO, Nprl3 +rapamycin and wild-type mice. (I) After PTZ injection, Nprl3 KO mice had a statistically significant average decrease (ANOVA; P < 0.05) in latency to seizure (77.2 s) compared with wild-type mice (168 s). Rapamycin treated Nprl3 KO mice had seizure latencies that did not differ from control mice (178 s). ***P < 0.001, *P < 0.05. Calibration mark in A = 200 µM.

Article Snippet: Whole brains were post-fixed in 4% PFA overnight [12 h for postnatal Day 3 (P3) pups, 24 h for adult mice], paraffin embedded, microtome sectioned at 8 μm and probed with CTIP2 (1:500; rat D ow nloaded from https://academ ic.oup.com /brain/article/145/11/3872/6524443 by guest on 30 August 2024 monoclonal; Abcam), GFP (1:1000; chicken polyclonal; Abcam), MAP2 (1:100; goat polyclonal; Abcam), PS6 (240/244) or ribosomal S6 protein (mouse monoclonals; 1:2000; Cell Signaling) primary antibodies and then secondary antibodies Alexa488, 647 and 594, respectively.

Techniques: In Vivo, Control, In Utero, Electroporation, Injection

Journal: The Journal of Neuroscience

Article Title: Cenpj Regulates Cilia Disassembly and Neurogenesis in the Developing Mouse Cortex

doi: 10.1523/JNEUROSCI.1849-18.2018

Figure Lengend Snippet: Cenpj was specifically depleted in the dorsal telencephalon of CenpjCKO mice. A, Schematic of Cenpj-trapped mice construction strategy. A LacZ reporter cassette and a neo tag, flanked by two FRT sequences, were introduced between the fourth exon and the fifth exon of Cenpj gene locus. The fifth exons were flanked by two LoxP sites in CenpjLacZ mice. LacZ reporter cassettes can be removed by Flippase splicing and the Cenpj gene can be knocked out by tissue-specific Cre recombinase splicing. B, Western blot analysis for Cenpj +/+ and CenpjCKO cerebral cortex lysate at E15.5. C, Quantification of Cenpj knock-out by Western blot. Histogram shows the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3. D, Brain sections were immunostained by anti-Pericentrin (green) antibody and anti-Cenpj (red) antibody on the ventricular surface in Cenpj+/+ and CenpjCKO mice. Scale bar, 10 μm. E, Representative whole-mount images of Cenpj+/+ and CenpjCKO brains at E15.5. Scale bar, 0.5 mm. F, Quantification of cerebral hemisphere circumference at E15.5. Data are presented as the mean ± SD; ***p = 0.0004 as determined by a t test; n = 3. G, Representative images of Cenpj+/+ and CenpjCKO brain coronal sections at E15.5 by Nissl staining. Right, Magnified cortical column. Scale bars, 500 μm (left) and 100 μm (right). H, Histogram of the cerebral cortex thickness. Data are presented as the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3; 3 brain slices per experiment. I, Body size of Cenpj+/+ and CenpjCKO mice at P7. J, Representative images of the Cenpj+/+ and CenpjCKO brain size at P7. Scale bar, 1 mm. K, Quantification of P7 cerebral hemisphere circumference. Data are presented as the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3. L, Representative images of Ctip2 (green) and Satb2 (red) expressions in the Cenpj+/+ and CenpjCKO cortex at P7. High-magnification images show the cortical column. Scale bar, 30 μm. M, Quantification of Ctip2+ cells and Satb2+ cells ratios in the neocortex. Data are presented as the mean ± SD; *pCtip2 = 0.0335, **pSatb2 = 0.0011 as determined by a t test; n = 3; 3 brain slices per experiment. N, Representative images of Foxp2 and Cux1 expressions in the Cenpj+/+ and CenpjCKO cortex at P7. Scale bar, 30 μm. O, Quantification of Foxp2+ cells ratios in the neocortex. Data are presented as the mean ± SD; *p = 0.0175, as determined by a t test; n = 3; 3 brain slices per experiment. P, Quantification of Cux1+ cells ratios in the neocortex. Data are presented as the mean ± SD; ***p = 0.0001 as determined by a t test; n = 3; 3 brain slices per experiment.

Article Snippet: Primary antibodies used were as follows: Zo-1 (1:300, 339100; Invitrogen), γ-Tubulin (1:5000, T3559; Sigma-Aldrich), γ-Tubulin (1:5000, T5326; Sigma-Aldrich), Acetylated Tubulin (1:30000, T7451; Sigma-Aldrich), Cenpj (1:50, 11517-1-AP; Proteintech), Pericentrin (1:300, 611814; BD Biosciences), GFP (1:500, GFP-1020; Aves Laboratories), Cleaved Caspase 3 (1:300, 9664; Cell Signaling Technology), Tbr2 (1:300, ab23345; Abcam), Satb2 (1:300, ab34735; Abcam), Ctip2 (1:300, ab18465; Abcam), Ki67 (1:300, ab9260; Millipore), BrdU (1:300, ab6326; Abcam), Pax6 (1:300, PRB-278P; Covance), Sox2 (1:300, sc-17320; Santa Cruz Biotechnology), EdU (Click-iT EdU Alexa Fluor 594 Imaging Kit, {"type":"entrez-nucleotide","attrs":{"text":"C10339","term_id":"1535410","term_text":"C10339"}} C10339 ; Thermo Scientific), GFAP (1:500, G9269,Sigma-Aldrich), and β-Catenin (1:300, 610153; BD Biosciences).

Techniques: Western Blot, Knock-Out, Staining

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal α-CtIP (D76F7) , Cell Signaling Technology, USA , Cat#9201; RRID: AB_10828593.

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis