Journal: bioRxiv

Article Title: Short and long TNF-alpha exposure recapitulates canonical astrogliosis events in human induced pluripotent stem cells-derived astrocytes

doi: 10.1101/722017

Figure Lengend Snippet: Experimental Design. (a) Derivation of human iPSC-astrocytes. Human neural stem cells (NSC) were differentiated from induced pluripotent stem cells (iPSC) and they were exposed to astrocyte induction medium (AIM). After 21 days in AIM human iPSC-derived radial glia-like cells (RCG) exhibited high proliferation rates and strong labeling for neural precursor markers. After exposing RGC to astrocyte medium for additional 4 weeks, human iPSC-derived astrocytes were obtained and used in the experiments. (b) Induction of in vitro astrogliosis. Human iPSC-derived astrocytes were exposed to serum-deprived culture medium for 24 h; then TNF-α was added to the cells in order to analyze the canonical events over the course of astrogliosis, as follows: i) NF-kB nuclear translocation was assessed 1 h after TNF-α exposure; ii) expression and secretion of cytokines and D-aspartate uptake assay were performed 24 h following TNF-α exposure; iii) densitometry of the intermediate filaments, vimentin and GFAP, cell morphology and viability, D-aspartate uptake assays were performed 5 days after TNF-α stimulation in order to investigate chronic stage of astrogliosis.

Article Snippet: We have used the following antibodies in this study: mouse anti-NF-kB p65 (1:100; sc-8008 - Santa Cruz Biotechnology, TX, USA), rabbit anti-Vimentin (1:2000; ab92547 – abcam, Cambridge, UK), mouse anti-GFAP (1:200; MO15052 – Neuromics, MN, USA).

Techniques: Derivative Assay, Labeling, In Vitro, Translocation Assay, Expressing

Journal: bioRxiv

Article Title: Short and long TNF-alpha exposure recapitulates canonical astrogliosis events in human induced pluripotent stem cells-derived astrocytes

doi: 10.1101/722017

Figure Lengend Snippet: Morphological analysis of human iPSC-derived activated astrocytes following 5 days TNF-α stimulation. (a) - Photomicrographs of human iPSC-derived astrocytes immunostained for vimentin (red), GFAP (green) and DAPI (blue); (b) – Quantification of vimentin immunostaining; (c) – Quantification of cell area in vimentin-stained cells. (d) – Percentage of astrocyte polarization, which cells were classified according to increase of length/width ratio of vimentin-stained labeling. (e) - Quantification of GFAP immunostaining expressed in arbitrary units of immunofluorescence (A.U); (f) - Quantification of DAPI-stained nuclei areas expressed as percentage of micrometers. Data are presented as means ± SEM from 4 cell lines in experiments performed in triplicates. **P < 0.01. Unpaired Student’s t-test. Photomicrographs magnification: 200x. Calibration bar: 100 μm.

Article Snippet: We have used the following antibodies in this study: mouse anti-NF-kB p65 (1:100; sc-8008 - Santa Cruz Biotechnology, TX, USA), rabbit anti-Vimentin (1:2000; ab92547 – abcam, Cambridge, UK), mouse anti-GFAP (1:200; MO15052 – Neuromics, MN, USA).

Techniques: Derivative Assay, Immunostaining, Staining, Labeling, Immunofluorescence

Journal: Nutrition & Diabetes

Article Title: Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice

doi: 10.1038/nutd.2012.10

Figure Lengend Snippet: Lack of change in acellular capillary, retinal ganglion cell and photoreceptor densities in db/db mice on ω-3 or ω-6PUFA feeds. Representative images of retinal capillaries from ω-3- ( a ) or ω-6PUFA-fed ( b ) db/db mice at 26 weeks of age reveal no statistically significant change in the number of acellular capillaries (white arrows) as quantified in ( c ). Representative images of β-III tubulin-stained RGCs from the peripheral ( d ) and central regions ( e ) of retinas from ω-3- and ω-6PUFA-fed db/db mice at 26 weeks reveal no statistically significant change in total numbers between feed groups ( f ). ( g ) Real-time qPCR quantification of β-III tubulin from whole retinas of 26-week-old db/db mice (normalized to millions of copies of Cyclophilin A (cyclo) mRNA) confirm comparable numbers of RGCs in both feeds; n =7 retinas for ω-3PUFA and n =5 for ω-6PUFA group. Photoreceptor densities were unaffected by feeds. 4',6-diamidino-2-phenylindole (DAPI)-stained retinas from ω-3- ( h ) and ω-6-fed ( i ) db/db mice at 26 weeks of age revealed that photoreceptor densities were unaffected. Real-time qPCR quantification of cone opsin ( j ) and rhodopsin ( k ) mRNA.

Article Snippet: Ganglion cells were quantified in both central and peripheral regions of the retina in a masked fashion in 26-week-old db/db mouse retinas fixed in 4% paraformaldehyde, stained with β-III tubulin primary antibody (C terminus, TP1691, ECM Biosciences, Versailles, KY, USA), and then whole-mounted with the photoreceptor side down and embedded in SlowFade Antifade reagent (Invitrogen, Grand Island, NY, USA).

Techniques: Staining

Journal: PLoS ONE

Article Title: Memantine Attenuates Alzheimer’s Disease-Like Pathology and Cognitive Impairment

doi: 10.1371/journal.pone.0145441

Figure Lengend Snippet: A,B) As compared with AAV1-GFP rats, AAV1-I 1 PP2A rats showed a marked decrease in the expression of somatodendritic marker MAP2 and synaptic markers synapsin and synaptophysin in the hippocampus. Memantine treatment restored dendritic and synaptic loss. B) Western blots showed that β III tubulin staining was significantly decreased in the hippocampus of AAV1-I 1 PP2A rats as compared with AAV1-GFP control animals. Memantine restored the decrease to physiological level. C)Fluoro Jade staining showed a selective neurodegeneration in the AAV1-I 1 PP2A rat dentate gyrus of the hippocampus. Memantine prevented the neurodegeneration in the AAV1-I 1 PP2A rats. 3–4 rats were employed in each experiment. *P<0.05, **P<0.01 AAV1-I 1 PP2A vs. AAV1-GFP rats; Δ P<0.05, ΔΔ P<0.01 AAV1-I 1 PP2A -memantine vs. AAV1-I 1 PP2A -vehicle. Scale bar in A and D, 100 μm.

Article Snippet: The antibodies employed in the present study are listed as follows: The primary antibodies used were purified mouse anti-PP2A catalytic α (1:10000; BD Transduction Laboratories TM ), demethylated anti-PP2AC 4b7 (1:5000; Millipore), pan-tau antibody, rabbit polyclonal antibody (pAb) 92e (1:5000; [ ]), phosphospecific tau antibodies: Tau pAb pS199 (1:1000, BioSource, Camarillo, CA, USA), Tau pAb pT205 (1:1000; BioSource), Tau pAb pS214 (1:500; BioSource), Tau monoclonal antibody (mAb) M4 to phosphorylated Thr 231/Ser-235 (1:1500; [ ]), Tau mAb 12E8 to phosphorylated Ser 262/356 (1:500; [ ]), Tau pAb pS396 (1:1000; BioSource), Tau pAb R145 to pS422 (1:3000; [ ]), I 1 PP2A mAb (5G6, 8.4 μg/ml; generated against recombinant human I 1 PP2A ; unpublished), mAb GFP (1:1000; Rockland, Gilbertville, PA, USA), mAb β-actin (1:2000; Sigma), mAb DM1A to α-tubulin (1:1000; Sigma), and rabbit pAb βIII tubulin (1:5000; Covance, Princeton, NJ, USA).

Techniques: Expressing, Marker, Western Blot, Staining, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: Increased Expression of Autophagy-Related Genes in Alzheimer’s Disease—Type 2 Diabetes Mellitus Comorbidity Models in Cells

doi: 10.3390/ijerph20054540

Figure Lengend Snippet: Sqstm1 and β-Tubulin III immunoreactivity in hippocampal and cortical sections of wild-type or 3xTg-AD mice at 12 months; scale bar: 20 µm ( A ). Sample Sqstm1 immunoblot ( B ). Sqstm1 levels in 3xTg-AD (3xTg) or wild-type (wt) mice at 6, 12, and 18 months (mo) of age ( C ). The means + SEM are plotted; n = 6/group.

Article Snippet: In immunofluorescence experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam, Cambridge, UK, cat # ab211324) 1:100; anti-SQSTM1/p62 (Abcam, cat # ab56416) 1:50; anti-β-Tubulin III (Merck Millipore, Burlington, MA, USA cat # T2200) 1:500; anti-MAP2 (1:500, Merck Millipore, cat # M9942) 1:500; anti-GFAP (Thermo Fisher Scientific, Waltham, MA, USA, cat # 13-0300), 1:800, donkey anti-rabbit-IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat # R37118) 1:1000; donkey anti-mouse-IgG Alexa Fluor 594 (Thermo Fisher Scientific, cat # A-21203, 1:1000); goat anti-mouse IgG1 CF 568 (Merck, cat # SAB4600313 1:1000); goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, cat # A21247, 1:1000); and DAPI (4′,6-diamidino-2-phenylindole Merck Millipore, cat #D9542) 1:5000.

Techniques: Western Blot

Journal: Molecular Oncology

Article Title: Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth

doi: 10.1002/1878-0261.12800

Figure Lengend Snippet: Biological effects of ectopic expression of miR‐326 and ARRB1 in MB CSCs. (A–C) MB CSCs were assayed 48 h after transfection with miR‐326 and ARRB1 , individually or combined. Mock‐transfected cells served as controls. Ectopic expression of ARRB1 and/or miR‐326 in these cells (A) reduced proliferation (MTT assay) (24 h: vs. CTRL: miR‐326+ P = 0.0072; ARRB1‐HA+ P = 0.0245; miR‐326+ARRB1‐HA+ P = 0.0015; 48 h: vs. CTRL: miR‐326+ P = 0.001; ARRB1‐HA+ P = 0.0005; miR‐326+ARRB1‐HA+ P < 0.0001), (B) diminished the frequency of oncosphere‐forming cells (vs. miR‐326‐ARRB1‐HA−: miR‐326+ P = 0.001; ARRB1‐HA+ P = 0.0005; miR‐326+ARRB1‐HA+ P = 0.0003), (C) decreased NANOG expression and increased the expression of PARP‐C (miR‐326 levels: miR‐326+ P < 0.0001; miR‐326+ARRB1‐HA+ P = 0.0002 vs. miR‐326‐ARRB1‐HA−). (D) qRT‐PCR revealed significantly increased expression of neuronal and glial differentiation markers ( βIII tubulin and GFAP , respectively) only in MB CSCs overexpressing miR‐326 (alone or with ARRB1 ) (vs. Mock βIII tubulin: miR‐326 P = 0.0027; miR‐326 and ARRB1 P = 0.046, GFAP: miR‐326 P = 0.0002; miR‐326 and ARRB1 P = 0.011). Data represent means ± SD from five independent experiments. Statistics: One‐way ANOVA and two‐way ANOVA, * P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001 vs. indicated controls.

Article Snippet: Cell was stained with mouse monoclonal antibodies against βIII tubulin (MAB 1637; Merck, Darmstadt, Germany) and GFAP (MAB360).

Techniques: Expressing, Transfection, MTT Assay, Quantitative RT-PCR

Journal: Frontiers in Neuroscience

Article Title: A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells

doi: 10.3389/fnins.2020.00607

Figure Lengend Snippet: Descriptions of the antibodies used for NSPC characterization including antibody specificity, dilution used, and source.

Article Snippet: β-iii tubulin , Cytoskeletal protein abundant in neural precursors , 1:500 , STEMCELL Technologies.

Techniques: Membrane, Marker

Journal: Frontiers in Neuroscience

Article Title: A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells

doi: 10.3389/fnins.2020.00607

Figure Lengend Snippet: Human, porcine, and rodent primary NSPCs grew in EFH proliferate (BrdU + ) and express neural stem cell marker Sox2. Upon differentiation, NSPCs are multipotent and generate β-iii tubulin + neural precursors, GFAP + astrocytes, and O4 + oligodendrocytes. No O4 + staining was observed from porcine NSPCs. Scale bar = 100 μm.

Article Snippet: β-iii tubulin , Cytoskeletal protein abundant in neural precursors , 1:500 , STEMCELL Technologies.

Techniques: Marker, Staining