Journal: Science Advances

Article Title: CHI3L1/YKL-40 signaling inhibits neurogenesis in models of Alzheimer’s disease

doi: 10.1126/sciadv.adv1492

Figure Lengend Snippet: ( A ) Immunoblots of 17 candidate mediators after 10-min CHI3L1 exposure (0 to 500 ng/ml). Phospho/total signals were densitometrically normalized to the no-CHI3L1 control (1.0); n = 3 to 4. ( B ) Dose-response blots for p-S6K1 and p-S6 (10 min). Values are phospho/total, expressed relative to untreated cells (1.0); n = 4. ( C ) Dose-response blots for p-IKKβ with the same quantification as in (B); n = 3. ( D ) BMS345541 (10 μM, 30 min) was added 10 min before CHI3L1 (500 ng/ml). p-S6 was quantified as in (B); n = 4. ( E ) iNPCs transfected with CRTH2 siRNA (siCRTH2) or control siRNA (siNC) were exposed to CHI3L1 (500 ng/ml, 48 hours). Proliferation = EdU + /DAPI + cells (% of total DAPI + ); n = 4. ( F ) After the same transfection, cells were differentiated for 3 days under CHI3L1 incubation. Neuronal differentiation output = DCX + /DAPI + (%), expressed as fold change over siNC+Ctrl (1.0); n = 4. Statistics: one-way ANOVA with Tukey’s post hoc for [(A) to (C)] and (E); unpaired t test for (D). * P < 0.05; ** P < 0.005, *** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: To determine the requirement of CRTH2 in the CHI3L1 signaling pathway, we performed RNAi-mediated knockdown of CRTH2 in iNPCs using a validated siRNA targeting human CRTH2 (siCRTH2; Santa Cruz Biotechnology, #sc-39838) or a control siRNA (siCtrl; Santa Cruz Biotechnology, #sc-37007).

Techniques: Western Blot, Control, Transfection, Incubation

Journal: Science Advances

Article Title: CHI3L1/YKL-40 signaling inhibits neurogenesis in models of Alzheimer’s disease

doi: 10.1126/sciadv.adv1492

Figure Lengend Snippet: ( A ) Design and timeline. Lentiviruses expressing shCRTH2-eGFP or shCtrl-eGFP were injected bilaterally into the DG of 6-month-old wild-type (WT) and 5XFAD mice (shCtrl in one side and shCRTH2 in the other). BrdU was given immediately, EdU 24 hours before euthanasia 4 weeks later. Groups: WT + shCtrl, 5XFAD + shCtrl, and 5XFAD + shCRTH2 ( n = 5). ( B ) Thioflavin-S/Aβ 42 staining showed hippocampal plaques in 5XFAD but not WT; CRTH2 knockdown did not change plaque size or density. ( C ) GFAP and CHI3L1 Immunolabeling indicated comparable astrocytosis and CHI3L1 induction in 5XFAD hemispheres with or without shCRTH2. ( D ) SGZ proliferation. EdU+ GFP+ cells were classified as radial NSCs (GFAP+) or amplifying progenitors (GFAP−). Proliferation was reduced in 5XFAD + shCtrl versus WT but significantly restored by shCRTH2. ( E ) Neuronal output. GFP+ BrdU+ cells coexpressing DCX (immature) or NeuN (mature) were decreased in 5XFAD + shCtrl and rescued by CRTH2 knockdown. Scale bars, 100 μm. ** P < 0.005, *** P < 0.001, and **** P < 0.0001 by one-way ANOVA with Tukey’s post hoc (comparisons versus WT + shCtrl and 5XFAD + shCtrl).

Article Snippet: To determine the requirement of CRTH2 in the CHI3L1 signaling pathway, we performed RNAi-mediated knockdown of CRTH2 in iNPCs using a validated siRNA targeting human CRTH2 (siCRTH2; Santa Cruz Biotechnology, #sc-39838) or a control siRNA (siCtrl; Santa Cruz Biotechnology, #sc-37007).

Techniques: Expressing, Injection, Staining, Knockdown, Immunolabeling

Journal: EJNMMI research

Article Title: The development of a GPR44 targeting radioligand [ 11 C]AZ12204657 for in vivo assessment of beta cell mass.

doi: 10.1186/s13550-018-0465-6

Figure Lengend Snippet: Fig. 4 In vitro autoradiography of [11C]AZ12204657 reveals focal binding patterns on pancreatic sections from non-diabetic human donors (a). The binding could be competed away with an excess of GPR44 antagonist AZD3825 (b). Similar GPR44-mediated binding was seen in pancreatic sections from T2D donors (c, d). The focal binding corresponded to Islets of Langerhans as assessed by immunofluorescent insulin staining (e, f) (representative results from three independent experiments on human sections with measurements performed in duplicates). The islet specific targeting was further assessed by [11C]AZ12204657 binding to homogenates of purified human islets of Langerhans and exocrine tissue preparations (results are from two independent experiments on homogenates with measurements performed in triplicates) (g). Binding of [11C]AZ12204657 in pancreatic sections from NHP was similarly focal in nature (h), consistent with the heterogeneous distribution of islets of Langerhans (i), and GPR44-mediated (j) (representative results from six independent experiments on NHP sections with measurements performed in singlets or duplicates)

Article Snippet: Confocal microscopy of GPR44, insulin, and glucagon in human islets of Langerhans Briefly, human islets were stained with a polyclonal rabbit antibody against GPR44 (HPA014259, dilution 1:10, Atlas Antibodies, Stockholm, Sweden), as well as with rat anti-human insulin (MAB1417, dilution 1:100, R&D Systems, Minneapolis, MN, USA) and Alexa647-conjugated mouse anti-glucagon (G2654, dilution 1:500, Sigma-Aldrich).

Techniques: In Vitro, Autoradiography, Binding Assay, Staining, Purification

Journal: EJNMMI research

Article Title: The development of a GPR44 targeting radioligand [ 11 C]AZ12204657 for in vivo assessment of beta cell mass.

doi: 10.1186/s13550-018-0465-6

Figure Lengend Snippet: Fig. 3 In vitro ligand binding to membranes of HEK293 cells transfected with human recombinant GPR44 and ligand potency at human GPR44 using a clonal beta cell line (EndoC-βH1). [3H]ProstaglandinD2 (a) and [6-3H-phenoxy]-AZ12204657 (b) binding to human GPR44 was displaced with increasing concentrations of AZ12204657. Results are from two independent experiments with three measurements at each concentration and presented as mean ± SD. The potency of AZ12204657 to inhibit the signal of 15(R)-15-methyl-PGD2 in human beta cells measured by the label free DMR assay (c). Results are from two independent experiments with two measurements at each concentration and presented as mean ± SD

Article Snippet: Confocal microscopy of GPR44, insulin, and glucagon in human islets of Langerhans Briefly, human islets were stained with a polyclonal rabbit antibody against GPR44 (HPA014259, dilution 1:10, Atlas Antibodies, Stockholm, Sweden), as well as with rat anti-human insulin (MAB1417, dilution 1:100, R&D Systems, Minneapolis, MN, USA) and Alexa647-conjugated mouse anti-glucagon (G2654, dilution 1:500, Sigma-Aldrich).

Techniques: In Vitro, Ligand Binding Assay, Transfection, Recombinant, Binding Assay, Concentration Assay

Journal: EJNMMI research

Article Title: The development of a GPR44 targeting radioligand [ 11 C]AZ12204657 for in vivo assessment of beta cell mass.

doi: 10.1186/s13550-018-0465-6

Figure Lengend Snippet: Fig. 5 Confocal microscopy of a human islet, showing antibody staining for cell nucleus (blue, a), insulin (green, b), GPR44 (red, c) and Glucagon (purple, d). Co-staining between insulin and GPR44 shows up in yellow in the composite image (e)

Article Snippet: Confocal microscopy of GPR44, insulin, and glucagon in human islets of Langerhans Briefly, human islets were stained with a polyclonal rabbit antibody against GPR44 (HPA014259, dilution 1:10, Atlas Antibodies, Stockholm, Sweden), as well as with rat anti-human insulin (MAB1417, dilution 1:100, R&D Systems, Minneapolis, MN, USA) and Alexa647-conjugated mouse anti-glucagon (G2654, dilution 1:500, Sigma-Aldrich).

Techniques: Confocal Microscopy, Staining

Journal: Science Advances

Article Title: CHI3L1/YKL-40 signaling inhibits neurogenesis in models of Alzheimer’s disease

doi: 10.1126/sciadv.adv1492

Figure Lengend Snippet: ( A ) Immunoblots of 17 candidate mediators after 10-min CHI3L1 exposure (0 to 500 ng/ml). Phospho/total signals were densitometrically normalized to the no-CHI3L1 control (1.0); n = 3 to 4. ( B ) Dose-response blots for p-S6K1 and p-S6 (10 min). Values are phospho/total, expressed relative to untreated cells (1.0); n = 4. ( C ) Dose-response blots for p-IKKβ with the same quantification as in (B); n = 3. ( D ) BMS345541 (10 μM, 30 min) was added 10 min before CHI3L1 (500 ng/ml). p-S6 was quantified as in (B); n = 4. ( E ) iNPCs transfected with CRTH2 siRNA (siCRTH2) or control siRNA (siNC) were exposed to CHI3L1 (500 ng/ml, 48 hours). Proliferation = EdU + /DAPI + cells (% of total DAPI + ); n = 4. ( F ) After the same transfection, cells were differentiated for 3 days under CHI3L1 incubation. Neuronal differentiation output = DCX + /DAPI + (%), expressed as fold change over siNC+Ctrl (1.0); n = 4. Statistics: one-way ANOVA with Tukey’s post hoc for [(A) to (C)] and (E); unpaired t test for (D). * P < 0.05; ** P < 0.005, *** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: To determine the requirement of CRTH2 in the CHI3L1 signaling pathway, we performed RNAi-mediated knockdown of CRTH2 in iNPCs using a validated siRNA targeting human CRTH2 (siCRTH2; Santa Cruz Biotechnology, #sc-39838) or a control siRNA (siCtrl; Santa Cruz Biotechnology, #sc-37007).

Techniques: Western Blot, Control, Transfection, Incubation

Journal: Science Advances

Article Title: CHI3L1/YKL-40 signaling inhibits neurogenesis in models of Alzheimer’s disease

doi: 10.1126/sciadv.adv1492

Figure Lengend Snippet: ( A ) Design and timeline. Lentiviruses expressing shCRTH2-eGFP or shCtrl-eGFP were injected bilaterally into the DG of 6-month-old wild-type (WT) and 5XFAD mice (shCtrl in one side and shCRTH2 in the other). BrdU was given immediately, EdU 24 hours before euthanasia 4 weeks later. Groups: WT + shCtrl, 5XFAD + shCtrl, and 5XFAD + shCRTH2 ( n = 5). ( B ) Thioflavin-S/Aβ 42 staining showed hippocampal plaques in 5XFAD but not WT; CRTH2 knockdown did not change plaque size or density. ( C ) GFAP and CHI3L1 Immunolabeling indicated comparable astrocytosis and CHI3L1 induction in 5XFAD hemispheres with or without shCRTH2. ( D ) SGZ proliferation. EdU+ GFP+ cells were classified as radial NSCs (GFAP+) or amplifying progenitors (GFAP−). Proliferation was reduced in 5XFAD + shCtrl versus WT but significantly restored by shCRTH2. ( E ) Neuronal output. GFP+ BrdU+ cells coexpressing DCX (immature) or NeuN (mature) were decreased in 5XFAD + shCtrl and rescued by CRTH2 knockdown. Scale bars, 100 μm. ** P < 0.005, *** P < 0.001, and **** P < 0.0001 by one-way ANOVA with Tukey’s post hoc (comparisons versus WT + shCtrl and 5XFAD + shCtrl).

Article Snippet: To determine the requirement of CRTH2 in the CHI3L1 signaling pathway, we performed RNAi-mediated knockdown of CRTH2 in iNPCs using a validated siRNA targeting human CRTH2 (siCRTH2; Santa Cruz Biotechnology, #sc-39838) or a control siRNA (siCtrl; Santa Cruz Biotechnology, #sc-37007).

Techniques: Expressing, Injection, Staining, Knockdown, Immunolabeling