Journal: Current Research in Toxicology

Article Title: Neural organoids incorporating microglia to assess neuroinflammation and toxicities induced by known developmental neurotoxins ☆

doi: 10.1016/j.crtox.2025.100252

Figure Lengend Snippet: Generation and immunofluorescence characterization of planar neuroimmune organoid. Organoids are generated in 96-well plates by the addition of iPSC-derived cells to the top of a 1) PEG-based hydrogel. Neuroimmune organoids were created by sequentially adding 2) neural progenitor cells (NPCs), 3) endothelial cells (ECs) and mesenchymal cells (MSCs), and finally 4) microglia (MGs). Organoids are cultured 21–28 days from initial NPC plating prior to treatments. For the neurotoxicity assessment herein, organoids were treated on day 23 and dosing occurred for 4 days. Cell culture supernatants were collected each day. On day 4, organoids were either harvested for transcriptional analysis or fixed for immunocytochemistry analysis. Representative images show the glial components of the neuroimmune organoids. Astrocytes and microglia were stained using antibodies to detect GFAP (green, left) or IBA1 (orange, right), respectively. Neuronal staining was performed using β3 tubulin (blue, both panels). The images shown are maximum intensity projections of 20X confocal images.

Article Snippet: Donkey anti-rabbit AF555 antibody (1:500, Thermo A31572), and AF-647 conjugated anti β3 tubulin antibody (1:250, RnD Systems IC1195R), was incubated overnight at 4 °C and washed three times in PBS.

Techniques: Immunofluorescence, Generated, Derivative Assay, Cell Culture, Immunocytochemistry, Staining

Journal: PLOS Genetics

Article Title: Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC

doi: 10.1371/journal.pgen.1010493

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Rabbit Tubulin , Novus Biologicals- NB100-56459 , 1:1000 WB.

Techniques:

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Mitochondrial ROS control neuronal excitability and cell fate in frontotemporal dementia.

doi: 10.1002/alz.12394

Figure Lengend Snippet: FIGURE 6 Mitochondrial antioxidants reduce the surface levels of specific α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) subunits elevated in patients’ neurons. A,B, Redox proteomics analysis of oxidized peptides in patients (P) and control (C) groups with and without MitoQ treatment (100 nM). A, Histograms represent the mean ± standard deviation of the number of oxidized peptides in control n = 4, c+mitoQ n = 2, patients n = 6, patients+mitoQ, n = 3 preparations. ** P < 0.01, two-way analysis of variance. B, Specific proteins showing a reduction in the number of oxidized peptides after the treatment with MitoQ (100 nM) in patients as analyzed by redox proteomics. Only proteins that showed a reduction in the three samples investigated are shown. C-N, Intracellular and surface distribution of different AMPAR and NMDAR subunits analyzed by western blot after the crosslinking of the superficial receptors using BS3. E, H, K, N, Representative western blots showing the bands corresponding to the intracellular and surface (crosslinked) subunits of: (E) all the AMPAR subunits GluA(1-4), detected with a pan-AMPA antibody, (H) AMPAR subunit GluA1, (K) NMDAR subunit NR1, (N) NMDAR subunit NR2B. Neuron-specific βIII tubulin was used as a loading control. Absence of higher molecular weight bands in the actin band (E) confirms that intracellular proteins were not crosslinked. Histograms show the quantification of the number of samples indicated in brackets, lines represent the median. For the quantification of the total levels, intracellular and surface levels were added. In the total levels group, some additional experiments using non-crosslinked samples were also included. In all cases, data was normalized to control for each experiment. Statistical significance between control and patients was analyzed with the non-parametric Mann-Whitney test in all cases except (G), which followed a normal distribution and two-sample t-test was used, number of samples analyzed is indicated in brackets. Specific samples, as indicated by the dotted lines, were treated with the mitochondrial antioxidant MitoTEMPO (MT, 100 nM, 1h). Statistical significance of the effect of MitoTEMPO was analyzed with the non-parametric paired Wilcoxon signed-rank test, except in (G) where the paired t-test was used. Number of samples analyzed is indicated in the MT column

Article Snippet: Membranes were incubated overnight with the corresponding primary antibodies diluted in 5% bovine albumin serum: pan AMPA (mabn832) 1:1000 from Merck Millipore; NR1 (SAB4501301) 1:1000 from Sigma-Aldrich; GluA1 (ab109450) 1:2000 and NR2B (ab28373) 1:500 from Abcam; beta-actin (4970) 1:5000 from Cell Signaling Technologies; beta-III Tubulin (MAB1195) 1:5000 from R&D Systems; and afterward with the corresponding speciespecific horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Control, Standard Deviation, Western Blot, Molecular Weight, MANN-WHITNEY

Journal: Neurobiology of disease

Article Title: Mild guanidinoacetate increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain cell development.

doi: 10.1016/j.nbd.2015.03.029

Figure Lengend Snippet: Fig. 1. AAV2-transduced GAMT deficiency in 3D organotypic developing brain cell cultures. (A) The three sequences selected for GAMT RNAi, with their respective location on the rat Gamt gene ORF. Ctrl-2 mismatched and scrambled are the GAMT-2 siRNA-derived negative controls. (B) Dual luciferase assay for selection of the best GAMT shRNA. Co-transfection of ROC cells with pRNAT-CMV3.2/Neo/GAMT-1/2/3/empty and psiCHECK-GAMT. GAMT-2 shRNA led to the best knockdown. (C) GAMT protein expression in ROC cell monolayer after pRNAT- CMV3.2/Neo/GAMT-2 transfection or AAV2/GAMT-2 transduction (MOI 300). GAMT knockdown specificity was tested by transfection with empty vector or the negative controls pRNAT-CMV.3.2/Neo/mismatched/scrambled. GAMT-2 shRNA drove a specific GAMT protein knockdown, and AAV2 viruses efficiently transduced the RNAi effect. Western blotting experiment normalized with β-tubulin. (D) GAMT protein expression in 3D organotypic brain cell cultures at DIV8 (top), DIV18 (middle) and DIV28 (bottom) after scAAV2/GAMT-2 transduction (MOIs 100/300/1000). Mismatched and scrambled negative controls were used to evaluate RNAi specificity (MOI 1000). Western blotting experiments normalized with actin. (E) Single point comparative expression of GAMT protein at DIV8, 18 and 28 in a representative culture transduced by scAAV2/GAMT-2 (MOI 1000). The highest GAMT ex- pression and the strongest GAMT knockdown were observed at DIV18 (MOI 1000, n = 1). Western blotting experiment normalized with actin. Western blotting data presented in B,C,D: mean ± s.e.m. (n = 6 in 1 out of 5 representative experiments). *P b 0.05, **P b 0.01, ***P b 0.001.

Article Snippet: All the other primary antibodies used in this study are commercially available, as follows: GalC (1:100, MAB5254), GFAP (1:100/1:5000, MAB 360), MAP-2 (1:100, MAB378), NeuN (1:100, MAB377), p-NFM (clone NN18, 1:100, MAB5254) and GABAAR (1:100/1:500, MABN489, targeting the α1 subunit of GABAAR) mouse monoclonal and GAP43 (1:100/1:20,000, AB5220) rabbit polyclonal (Chemicon); β-tubulin (1:1000, T8578) mouse monoclonal (Sigma-Aldrich); Actin (1:1000, sc1616), MBP (1:500, sc13914) and GAD (1:100/1:500, sc7513) goat polyclonal; NFM (clone NF-09, 1:1000, sc51683) mouse monoclonal (Santa Cruz Biotechnology); cleaved caspase-3 (1:200, 9664) and caspase-3 (1:1000, 9665) rabbitmonoclonal (Cell Signaling Technology).

Techniques: Derivative Assay, Luciferase, Selection, shRNA, Cotransfection, Knockdown, Expressing, Transfection, Transduction, Plasmid Preparation, Western Blot