Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet: Sema5A increases enteric neurons synapsin 1 phosphorylation at site Ser-603 Western blot analysis of synapsin 1 phosphorylation at site Ser603, Ser9 and Ser62/67 from enteric neuron cultures exposed for 3 days to control Fc, Sema5A-Fc, or Sema5A-S956G-Fc. ∗ p < 0.05 Kruskal-Wallis test, supplemented by Dunn’s post-hoc test. Data are mean ± SEM, n = 8 cultures from 2 independent experiments.

Article Snippet: Membranes were incubated for 1h in blocking solution composed of Tris-buffered saline (TBS), 0.1% (v/v) Tween-20 and 5% (w/v) non-fat powder milk and were then incubated overnight at 4°C in primary antibodies as follows: rabbit polyclonal anti-Sema5A (respectively, 1:500, cat: NBP2-20291, Novus Biologicals and, 1:200, cat: 4460-MG, R&D Systems), rabbit polyclonal anti-Plexin A1 (1:500, cat: APR-081, Alomone), goat polyclonal anti-Plexin A2 (1:500, cat: AF5486, R&D Systems), rabbit monoclonal anti-synapsin 1 (1:1000, cat: 5297, Cell Signaling), mouse monoclonal anti β-tubulin (1:1000, cat: T8660, Sigma-Aldrich), rabbit polyclonal anti-phospho-synapsin 1 (Ser62/67) (1:1000, cat: 11583501, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser603) (1:1000, cat: PA1-4604, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser9) (1:1000, cat: CST2311, Cell Signaling), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (1:1000, cat: 9102, Cell Signaling), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, cat: 9101, Cell Signaling), mouse monoclonal anti-CaMKII alpha (1:1000, cat: MA1-048, Invitrogen), rabbit monoclonal anti-phospho-CaMKII (Thr286) (D21E4) (1:1000, cat: 12716, Cell Signaling) and mouse monoclonal anti-β-Actin (1:5000, cat: A5441, Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet:

Article Snippet: Membranes were incubated for 1h in blocking solution composed of Tris-buffered saline (TBS), 0.1% (v/v) Tween-20 and 5% (w/v) non-fat powder milk and were then incubated overnight at 4°C in primary antibodies as follows: rabbit polyclonal anti-Sema5A (respectively, 1:500, cat: NBP2-20291, Novus Biologicals and, 1:200, cat: 4460-MG, R&D Systems), rabbit polyclonal anti-Plexin A1 (1:500, cat: APR-081, Alomone), goat polyclonal anti-Plexin A2 (1:500, cat: AF5486, R&D Systems), rabbit monoclonal anti-synapsin 1 (1:1000, cat: 5297, Cell Signaling), mouse monoclonal anti β-tubulin (1:1000, cat: T8660, Sigma-Aldrich), rabbit polyclonal anti-phospho-synapsin 1 (Ser62/67) (1:1000, cat: 11583501, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser603) (1:1000, cat: PA1-4604, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser9) (1:1000, cat: CST2311, Cell Signaling), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (1:1000, cat: 9102, Cell Signaling), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, cat: 9101, Cell Signaling), mouse monoclonal anti-CaMKII alpha (1:1000, cat: MA1-048, Invitrogen), rabbit monoclonal anti-phospho-CaMKII (Thr286) (D21E4) (1:1000, cat: 12716, Cell Signaling) and mouse monoclonal anti-β-Actin (1:5000, cat: A5441, Sigma-Aldrich).

Techniques: Recombinant, Western Blot, Mutagenesis, Bicinchoninic Acid Protein Assay, Software, Membrane

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: Primers for the qPCR.

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques:

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: (A) mRNA levels of MAP2 , SYN1 , and LEF1 were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. All Wnts promoted MAP2 expression, and Wnt7a induced the highest SYN1 expression. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. 1 p <0.05, 1″ p <0.01 (DMEM vs. all groups); 2 p <0.05, 2″ p <0.01 (NTs vs. all groups); 3 p <0.05, 3″ p <0.01 (Wnt1 vs. all groups); 4 p <0.05, 4″ p <0.01 (Wnt3a vs. all groups); 5 p <0.05, 5″ p <0.01 (Wnt5a vs. all groups); 6 p <0.05, 6″ p <0.01 (Wnt7a vs. all groups). (B) mRNA levels of ChAT , DBH , and LEF1 were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. Wnt1 had no effects on ChAT or DBH expressions, but Wnt7a significantly induced both genes. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. 1 p <0.05, 1″ p <0.01 (DMEM vs. all groups); 2 p <0.05, 2″ p <0.01 (NTs vs. all groups); 3 p <0.05, 3″ p <0.01 (Wnt1 vs. all groups); 4 p <0.05, 4″ p <0.01 (Wnt3a vs. all groups); 5 p <0.05, 5″ p <0.01 (Wnt5a vs. all groups); 6 p <0.05, 6″ p <0.01 (Wnt7a vs. all groups).

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: (A) Wnt7a groups were treated with Wnt7a (2 µg/ml) for 2 days, and the NTs groups were treated with NTs for 9 days. After NTs treatment for 1 week, Wnt7a (2 µg/ml) was added to the NTs+Wnt7a groups for 2 days. mRNA levels of LEF1 , SYN1 , BSN , and SYTG were examined in hMSCs, and levels were normalized to those in the NTs control (set to 1.0). Wnt7a induced mRNA expressions of SYN1 , BSN , and SYTG in NT-stimulated hMSCs, and this induction was related to upregulation of LEF1 . Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. * p <0.05, ** p <0.01 (NTs and Wnt7a vs. DMEM; NTs+Wnt7a vs. NTs). (B) LiCl groups were treated with LiCl (4 mM) for 2 days, and NTs groups were treated with NTs for 9 days. After NTs treatment for 1 week, LiCl (1 or 4 mM) was added to the NTs+LiCl groups for 2 days, mRNA levels of SYN1 and LEF1 were examined in hMSCs, and their levels were normalized to those in the DMEM control (set to 1.0). * p <0.05, ** p <0.01 (NTs and LiCl vs. DMEM; NTs+LiCl vs. NTs).

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques:

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: (A) p4 NT-treated hMSCs were stained with β-catenin (green). The NTs+Wnt7a groups were treated with NTs+Wnt7a for 24 h, and the NTs+lithium groups were treated with NTs+lithium for 24 h. 4,6-Diamidino-2-phenylindole (DAPI) (blue) was used as a counterstain. (B) p4 NT-treated hMSCs were immunoblotted with MAP2, SYN1, and GAPDH. The NT group was treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) p4 NT-treated hMSCs were stained with MAP2 (red), SYN1 (red), and β-catenin (green). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then NTs+Wnt7a or lithium for 7 days. DAPI (blue) was used as a counterstain. DMEM groups served as the control. The white bar represents 50 µm. (D) Percentages of MAP2-positive cells, SYN1-positive cells, and neurite-positive cells among all DAPI-positive cells. All data are presented as the mean ± SD. * p <0.05, ** p <0.01 (all vs. NTs). (E) Cell areas were calculated by β-catenin-positive cells from (B).

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques: Staining

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: (A) As described in " ", mRNA levels of SYN1 and LEF1 were examined by a qPCR. sFRP4 showed significant inhibition of SYN1 expression, and Frz5 blocking antibodies greatly inhibited gene expressions. Levels were normalized to those in NTs groups (set to 1.0). * p <0.05, ** p <0.01 (NTs+Wnt7a vs. all groups). (B) Percentages of inhibition calculated from (A). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques: Inhibition, Expressing, Blocking Assay

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: (A) mRNA levels of ChAT and DBH were examined in NT-induced hMSCs, and SP600125 (15 µM) and Wnt7a (2 µg/ml) or LiCl (4 mM) were added to NT-induced hMSCs at the same time. Levels were normalized to those in the NTs control (set to 1.0). Wnt7a, but not lithium, stimulated mRNA levels in NT-induced hMSCs, and SP600125 totally inhibited Wnt7a-induced ChAT and DBH expressions. Data are presented as the mean ± SD of one triplicate experiment that was representative of the three independent experiments. * p <0.05, ** p <0.01 (all vs. NTs). # p <0.05, ## p <0.01 (all vs. NTs+Wnt7a+SP600125). + p <0.05, ++ p <0.01 (all vs. NTs+LiCl+SP600125). (B) p4 NT-treated hMSCs were immunoblotted with ChAT, DBH, and GAPDH. The NT groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) Expression levels of MAP2 and SYN1 in NT-induced hMSCs with SP600125/Wnt7a or SP600125/LiCl are shown. SP600125 had no effect in MAP2 or SYN1 expression. Levels were normalized to those in NTs groups (set to 1.0). * p <0.05, ** p <0.01 (NTs vs. all groups). # p <0.05, ## p <0.01 (all vs. NTs+Wnt7a+SP600125). + p <0.05, ++ p <0.01 (all vs. NTs+LiCl+SP600125). (D) p4 NT-treated hMSCs were stained with ChAT (green) and DBH (red). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for the first 7 days and then with NTs+Wnt7a or lithium for the next 7 days. In inhibitory groups, SP600125 was added with Wnt7a or lithium in NT-induced hMSCs at the same time. DAPI (blue) was used as a counterstain. DMEM groups were used as controls. The white bar represents 50 µm. (E) Percentages of ChAT-positive cells and DBH-positive cells among all DAPI-positive cells calculated from (D). All data are presented as the mean ± SD. * p <0.05, ** p <0.01 (all vs. NTs). # p <0.05, ## p <0.01 (all vs. NTs+Wnt7a+SP600125). + p <0.05, ++ p <0.01 (all vs. NTs+LiCl+SP600125). (F) As described in " ", mRNA levels of ChAT and DBH were examined by a qPCR. Levels were normalized to those in NTs groups (set to 1.0). * p <0.05, ** p <0.01 (NTs+Wnt7a vs. all groups). (G) Percentages of inhibition calculated from (E). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques: Expressing, Staining, Inhibition

Journal: PLoS ONE

Article Title: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

doi: 10.1371/journal.pone.0104937

Figure Lengend Snippet: Accompanying NGF, BDNF, and RA, Wnt7a activated the canonical/β-catenin pathway via receptor Frz5 to promote neurogenesis and trigger synaptic marker (SYN1) expression. Furthermore, Wnt7a also triggered differentiations of cholinergic and dopaminergic neurons, but this effect was induced by another non-canonical/JNK pathway through Frz9 receptors. In this study, we showed that Wnt7a utilized two pathways to promote hMSC neurogenesis.

Article Snippet: hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,).

Techniques: Marker, Expressing