Journal: PLOS ONE

Article Title: Characterization of the atypical antipsychotic drug aripiprazole cytotoxicity in the neutrophil model cell line HL-60

doi: 10.1371/journal.pone.0318878

Figure Lengend Snippet: Dopamine receptor subtype agonists and antagonists used in this study.

Article Snippet: The following primary antibodies were used to detect specific dopamine receptor subtypes: PE mouse anti-human Dopamine D1 receptor (BioLegend), rabbit anti-dopamine D2R (extracellular) receptor (Alomone labs), rabbit anti-dopamine D3R receptor (MilliporeSigma), rabbit anti-dopamine D4R receptor (Invitrogen), and rabbit anti-dopamine D5R receptor (Invitrogen).

Techniques:

Journal: Science Advances

Article Title: CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation

doi: 10.1126/sciadv.adg8148

Figure Lengend Snippet: ( A ) Summary of CHI3L1 receptors and downstream signaling pathways. ( B ) Expression of CRTH2 receptor in NSCs (GFAP + Sox2 + ) but not immature (DCX + ) or mature (NeuN + ) neurons in adult hippocampus. Scale bars, 20 μm. ( C ) CRTH2 expression in cultured NSCs (Sox2 + Nestin + ). ( D ) Activation of GSK-3β (reduced inhibitory serine-9 phosphorylation) and inhibition of β-catenin (increased degradation) in NSC cultures treated with CHI3L1 (100 ng/ml). n = 3. ( E ) Lentiviral vectors to express two shRNAs for CRTH2 knockdown (Lenti-shCRTH2, shCRTH2_1, and shCRTH2_2) and a control shRNA (Lenti-shNC). ( F ) shRNA-mediated CRTH2 knockdown efficiency. n = 3. ( G ) CRTH2 knockdown effect on GSK-3β/β-catenin pathway activation by CHI3L1 (2-hour treatment). n = 3. ( H ) CRTH2 knockdown effect on NSC proliferation in shRNA-expressing cultures, with or without CHI3L1 for 3 days. n = 4. Scale bars, 100 μm. ( I ) CRTH2 knockdown effect on NSC differentiation into neurons (Tuj1 + ) and glia (GFAP + ) in shRNA-expressing cultures, with or without CHI3L1 for 3 days. n = 4. Scale bars, 100 μm. ( J ) Identified CHI3L1 signaling pathway inhibitory to neurogenesis: CHI3L1 binds to CRTH2 receptor and activates GSK-3β that in turn phosphorylates and destabilizes β-catenin and leads to reduced transcriptional activities for neurogenesis. ( K ) Timeline for assays of NSC proliferation and neuronal differentiation in msCtrl-IgG– or msAQP4-IgG–injected mice, receiving a potent GSK-3β inhibitor, TWS119 (30 mg/kg), intraperitoneally daily for 3 weeks. ( L to N ) Quantification of total proliferating cells (EdU + ), radial glia–like NSCs (EdU + GFAP + ), and transiently amplifying progenitor-like (EdU + GFAP − ) cells. n = 4. ( O to Q ) Quantification of proliferating cells (BrdU + ) at the time of hsAQP4-IgG injection, newborn immature neurons (BrdU + DCX + ), and mature neurons (BrdU + NeuN + ). n = 4. All bar graphs presented in means ± SEM and analyzed by one-way ANOVA and Tukey’s post hoc analyses. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were mouse anti-GFAP (1:1000, CST), rabbit anti-GFAP (1:1000, CST), chicken anti-GFAP (1:1000, Abcam), rabbit anti-NeuN (1:500, Abcam), mouse anti-DCX (1:200, Santa Cruz Biotechnology), rabbit anti-Sox2 (1:500, Abcam), mouse anti-Sox2 (1:500, Abcam), rat anti-BrdU (1:1000, Abcam), mouse anti-Tuj1 (1:1000, Abcam), rabbit anti–IIL-13Rα2 (1:500, CST), rabbit anti-CHI3L1 (1:500, Abcam), goat anti-CHI3L1 (1:500, R&D Systems), rabbit anti-CHI3L1 (1:500, Solarbio), rabbit anti-CRTH2 (1:500, Invitrogen), mouse anti-CRTH2 (1:500, Invitrogen), and mouse anti-TMEM219 (1:500, CST).

Techniques: Expressing, Cell Culture, Activation Assay, Inhibition, shRNA, Injection

Journal: Science Advances

Article Title: CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation

doi: 10.1126/sciadv.adg8148

Figure Lengend Snippet: ( A ) Schematic diagram of assays for shRNA-mediated CRTH2 knockdown effect on NSC proliferation and differentiation affected by msAQP4-IgG. The lentiviruses expressing GFP together with a scrambled nontargeting control shRNA (Lenti-shNC) or an shRNA targeting CRTH2 (Lenti-shCRTH2_1). Scale bars, 100 μm. ( B ) CRTH2 knockdown effect on NSC proliferation in DG, quantified by the numbers of proliferating cells expressing an shRNA (GFP + EdU + ) and radial glia–like NSCs (GFP + EdU + GFAP). n = 4 animals. Scale bars, 20 μm. ( C ) Quantification of shRNA-expressing (GFP + ) NSC (BrdU + ) differentiation into immature neurons (DCX + ) in DG. n = 4. Scale bars, 20 μm. ( D ) Quantification of NSC differentiation into mature neurons (GFP + BrdU + NeuN + ) in DG. n = 4. Scale bars, 20 μm. ( E ) Schematic diagram of in vitro assays for the effect of AZD1981, a selective CRTH2 antagonist, on neurogenesis suppressed by CHI3L1 signaling secondary to mini-pump infusion of msCtrl-IgG or msAQP4-IgG. ( F ) Quantification of NSC proliferation in DG, in the presence of CHI3L1 or PBS and with AZD1981 or DMSO. n = 4. Scale bars, 100 μm. ( G ) Quantification of NSC neuronal differentiation. n = 4. Scale bars, 100 μm. ( H ) Schematic diagram of in vivo assays for AZD1981 efficacy to rescue neurogenesis affected by msAQP4-IgG–induced CHI3L1 signaling. ( I and J ) Quantification of NSC proliferation by computing total proliferating cells (EdU + ) and radial glia–like NSCs (EdU + GFAP + Sox2 + ) in DG. n = 4. ( K and L ) Quantification of NSC proliferation into immature (BrdU + DCX + ) and mature (BrdU + NeuN + ) neurons. n = 4 animals. All quantitative data presented as bar graphs in means ± SEM and evaluated by one-way ANOVA and Tukey’s post hoc multiple comparisons. Nonsignificant comparisons are not identified. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were mouse anti-GFAP (1:1000, CST), rabbit anti-GFAP (1:1000, CST), chicken anti-GFAP (1:1000, Abcam), rabbit anti-NeuN (1:500, Abcam), mouse anti-DCX (1:200, Santa Cruz Biotechnology), rabbit anti-Sox2 (1:500, Abcam), mouse anti-Sox2 (1:500, Abcam), rat anti-BrdU (1:1000, Abcam), mouse anti-Tuj1 (1:1000, Abcam), rabbit anti–IIL-13Rα2 (1:500, CST), rabbit anti-CHI3L1 (1:500, Abcam), goat anti-CHI3L1 (1:500, R&D Systems), rabbit anti-CHI3L1 (1:500, Solarbio), rabbit anti-CRTH2 (1:500, Invitrogen), mouse anti-CRTH2 (1:500, Invitrogen), and mouse anti-TMEM219 (1:500, CST).

Techniques: shRNA, Expressing, In Vitro, In Vivo