Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal IgG anti-His Tag (AD1.1.10’)(AlexaFluor®647) , NOVUS Biologicals , Cat# NB100–64768AF647.

Techniques: Purification, Marker, Recombinant, Produced, Antibody Labeling, Cell Culture, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Dentate gyrus morphogenesis is regulated by β-CATENIN function in hem-derived fimbrial glia

doi: 10.1101/2022.09.15.508086

Figure Lengend Snippet: (A, B) TBR2+ and PAX6+ progenitors are seen in the 2 and 3ry matrix of the DMS at E14.5 and E16.5, in control brains (DMS, white arrow). In Lmx1aCre::β-Catenin LOF brains, these cells are diverted to the ectopic glial protrusion (yellow arrow); representative images are shown of sections taken from N=4 (E14.5), N=5 (E16.5) brains (biologically independent replicates) examined over 3 independent experiments. (C) In control brains, REELIN+ CR cells reside in the hippocampal fissure above LEF1+ DMS cells, whereas in β-Catenin LOF brains both these cell types are co-mingled in the ectopic protrusion (yellow arrowhead). (D) Quantification of the images in (A). (E-G) PROX1+ dentate granule cells localize to the dentate gyrus in controls, but are diverted to the ectopic protrusion in β-Catenin LOF brains (yellow arrowhead). The boxed region in C, E and F are shown in high magnification in the adjacent images. (H) FoxJ1Cre does not drive Ai9 reporter expression in the BLBP+ fimbrial scaffold (asterisk) but efficiently labels the REELIN+ CR cells (white arrowheads). At E18.5, PROX1+ dentate granule cells and TRP73+ CR cells appear normally positioned in both control and Foxj1Cre::β-Catenin LOF brains (I) Quantifications of the images in (H). D: N= 4 (E14.5); N=5 (E16.5); G: N=5 (E16.5 and E18.5); I: N=3 (E18.5) biologically independent replicates examined over 3 independent experiments. In and , image stitching was performed. Statistical tests (D, G & I): Two-tailed unpaired Mann Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001, ns if p-value> 0.05; P=0.9268 (E), P=0.000011 (G, E16.5 and E18.5 MZ), P=0.000084 (G, E16.5 ectopic site), P=0.000076 (G, E18.5 ectopic site), P=0.489428 (I, TRP73), and P>0.999999 (I, PROX1). All scale bars: 100 μm.

Article Snippet: The primary antibodies used were: LEF1 (Rabbit, 1:200 CST catalogue # C12A5), β-CATENIN (Mouse 1:200, BDbiosciences catalogue # 610153), β-CATENIN (Rabbit 1:50, CST catalogue # 8814), RFP (Rabbit, 1:200 Abcam catalogue # ab62341), RFP (Mouse, 1:200 Invitrogen catalogue # MA5-15257), TBR2 (Rabbit, 1:200 Abcam catalogue # ab23345), TBR2 (Rat, 1:200 Invitrogen catalogue # 14-4875-82), PAX6 (Rabbit, 1:500 Abcam catalogue # ab195045), PROX1 (Rabbit, 1:500 Millipore catalogue # ab5475), BLBP (Rabbit, 1:200 sigma catalogue # ABN14),TRP73 (Rabbit, 1:200 CST catalogue # 14620S), REELIN (Mouse, 1:200 Millipore catalogue # MAb5364), TTR (Rabbit, 1:75 Dako catalogue # A0002), GFAP (Rabbit, 1:200 Sigma catalogue # G9269), GFAP (Mouse, 1:200 Sigma catalogue # G3893), N-CADHERIN (Mouse, 1:200 BDbiosciences catalogue # 610920), NEUROD1 (Rabbit, 1:1000 Abcam catalogue # AB213725), Phospho HISTONE H3 (Rabbit, 1:200 CST catalogue # H0412), Ki67 (Rabbit, 1:200 Abcam catalogue # ab15580), SOX9 (Rabbit, 1:200 Abcam catalogue # ab185230), SOX2 (Mouse, 1:200 Invitrogen catalogue #MA1-014), NFIA (Rabbit, 1:500 Abcam catalogue # ab228897), NFIB (Rabbit, 1:500 Abcam catalogue # ab186738), ALDH1L1(Rabbit, 1:200 Abcam catalogue #ab87117), AQP4 (Rabbit, 1:200 CST catalogue # 59678).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, IF-P

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Deficiency of FABP7 Triggers Premature Neural Differentiation in Idiopathic Normocephalic Autism Organoids.

doi: 10.1002/advs.202406849

Figure Lengend Snippet: Figure 5. FABP7 regulates premature neural differentiation associated with MAPK signals in ASD organoids. A) Schematic of FABP7-EGFP encoded by lentivirus. B) Schematic of injecting lentivirus encoding FABP7 or vehicle alone into D20 organoids and then culturing for another 10 days for infection. C) Representative images of immunostaining for PAX6 and GFP in the D30 IMR90-4 and ASD3-28 organoids infected with FABP7 overexpression and control virus, respectively. Scale bar: 25 μm. D) Histograms showing the quantification of PAX6 + GFP + /GFP+ cells in the IMR90-4 + LV-NC, IMR90- 4 + LV-FABP7, ASD3-28 + LV-NC and ASD3-28 + LV-FABP7 groups. IMR90-4 + LV-NC: n = 30 organoids, IMR90-4 + LV-FABP7: n = 28 organoids, ASD3-28 + LV-NC: n = 30 organoids, ASD3-28 + LV-FABP7: n = 30 organoids, IMR90-4 + LV-NC versus IMR90-4 + LV-FABP7: ***p < 0.001, ASD3- 28 + LV-NC versus ASD3-28 + LV-FABP7: ***p < 0.001, IMR90-4 + LV-NC versus ASD3-28 + LV-FABP7: ns p = 0.89. Organoids from 3 independent biological replicate experiments were analyzed for each cell line. E) Representative images of immunostaining for TBR1 and GFP in the D30 IMR90-

Article Snippet: CTIP2 Rat IgG 1:500(IF) abcam ab18465 DCX Rabbit IgG 1:1000(IF) Cell Signaling 4604 FABP7 Rabbit IgG 1:2000(WB) 1:500(IP) Cell Signaling Technology 13347S FOXG1 Rabbit IgG 1:1000(IF) abcam ab18259 FOXP2 Rabbit IgG 1:1000(IF) abcam ab16046 GAPDH Mouse IgG 1:5000(WB) affinity T0004 GFP Chicken IgG 1:1000(IF) Millipore ab16901 GFP Rabbit IgG 1:1000(IF) Chemicon AB3080 KI67 Rabbit IgG 1:500(IF) Invitrogen 180191Z MAP4K2 Rabbit IgG 1:2000(WB) abcam ab184169 MEK1/2 Mouse IgG 1:1000(WB) Cell Signaling Technology 4694S NANOG Goat IgG 1:1000(IF) R&D Systems AF1997 NESTIN Goat IgG 1:1000(IF) Santa Cruz SC-21247 PAX6 Rabbit IgG 1:500(IF) convance PRB-278P PHH3 Rat IgG 1:1000(IF) Cell Signaling Technology 9706S PKCλ Mouse IgG 1:1000(IF) BD 610 207 p-MEK1/2 (S217/221) Rabbit IgG 1:1000(WB) Cell Signaling Technology 3958S PROX1 Rabbit IgG 1:300(IF) abcam ab101851 p-Vimentin Mouse IgG 1:1000(IF) MBL D076-3 SOX2 Goat IgG 1:500(IF) R&D Systems AF2018 TBR1 Rabbit IgG 1:500(IF) abcam ab31940 γ-tublin Mouse IgG 1:1000(IF) abcam ab11316 Alexa Fluor 488 Goat anti-Chicken IgG 1:1000(IF) Invitrogen A11039 Alexa Fluor 488 Donkey anti-Rat IgG 1:1000(IF) Invitrogen A21208 Alexa Fluor 488 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A21206 Alexa Fluor 488 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A21202 Alexa Fluor 488 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11055 Alexa Fluor 546 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A10040 Alexa Fluor 546 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A10036 Alexa Fluor 546 Goat anti-Rat IgG 1:1000(IF) Invitrogen A11081 Alexa Fluor 546 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11056 Alexa Fluor 647 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A31571 Alexa Fluor 647 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A31573 Alexa Fluor 647 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A21447 Hoechst332258 1:1000(IF) Invitrogen A1339 HRP Goat Anti-Mouse IgG 1:5000(WB) Multi Sciences 70-GAM0072 HRP Goat Anti-Rabbit IgG 1:5000(WB) Multi Sciences 70-GAR0072 was reverse transcribed into cDNA using a PrimeScriptTM RT reagent kit (TaKaRa) and prepared for qPCR using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific).The primers for qPCR used were as follows: FABP7 forward primer, 5′-TCTCACCTCCTTCCTTCTTCT-3′ and reverse primer, 5′-AGAACCTTG CCAGTGATGTATT-3′; MEK2 forward primer, 5′-CTCACCATCAACCCTAC CATC-3′, and reverse primer, 5′-GCAGGTCCACCAGGTTT-3′; GAPDH forward primer, 5′-TCGACAGTCAGCCGCATCTTCTTT-3′ and reverse primer, 5′-ACCAAATCCGTTGACTCCGACCTT-3′.

Techniques: Infection, Immunostaining, Over Expression, Control, Virus

Journal: The Journal of Biological Chemistry

Article Title: A Novel Suppressive Effect of Alcohol Dehydrogenase 5 in Neuronal Differentiation *

doi: 10.1074/jbc.C114.561860

Figure Lengend Snippet: ADH5 counteracted differentiation of human neural stem cells toward neurons. A, immunostaining of the neural progenitor markers Nestin, Sox2, Pax6, and Ki67 in hNSCs. Scale bar, 50 μm. B, scheme of differentiating hNSCs toward neurons. Briefly, hNSCs were transduced with lentiviral vectors (ADH5, ADH5(R115D), or empty vector) at day 0. At day 4, the transduced hNSCs subcultured in a 24-well microplate were induced to differentiation toward neurons. Immunofluorescence and qPCR were performed at day 40. C, qPCR analysis of ADH5 expression in hNSCs and their neuron derivatives. D, qPCR analysis of SOX2 and Nestin in hNSCs transduced with lentivirus expressing either EGFP (Vector) or ADH5. E, Transwell assay to analyze the relative number of migrated hNSCs transfected with lenti-EGFP or lenti-ADH5 (wt). F, immunofluorescence staining of differentiated neurons indicated in B in the absence or presence of ADH5 inhibitor C3. Scale bar, 50 μm. G, qPCR analysis of ADH5, MAP2, and GAD67, in the differentiated neurons indicated in B and F. H, qPCR analysis of synapsin as a marker of neuron maturation in the differentiated neurons indicated in B and F. I, qPCR analysis of glial fibrillary acidic protein (GFAP), α-smooth muscle actin (SMA), and α-fetoprotein (AFP) lineage-specific markers in the differentiated neurons indicated in B and F. J, scheme of differentiating hNSCs toward neurons in the presence of ADH5 inhibitor C3. Briefly, hNSCs subcultured in a 24-well microplate at day 0 were induced to differentiation toward neurons at day 3 by differentiation medium together with C3. Immunofluorescence and qPCR were performed at day 9. K, qPCR analysis of neuron-specific markers, MAP2 and GAD67, in the differentiated hNSCs treated with C3 for 6 days. All the values are shown as means ± S.E. n = 3. Single asterisk, p < 0.05; triple asterisk, p < 0.001. NS, not significant.

Article Snippet: The following antibodies were used: β-actin (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), ADH5 (Proteintech), Sox2 (Abcam), Pax6 (Vector Laboratories), Nestin (Millipore), Ki67 (Covance), MAP2 (Millipore), and Tuj1 (Sigma-Aldrich).

Techniques: Immunostaining, Transduction, Plasmid Preparation, Immunofluorescence, Expressing, Transwell Assay, Transfection, Staining, Marker