Journal: Cell reports methods

Article Title: A versatile vessel casting method for fine mapping of vascular networks using a hydrogel-based lipophilic dye solution.

doi: 10.1016/j.crmeth.2023.100407

Figure Lengend Snippet: Figure 4. Compatibility of VALID with 2D and 3D immunolabeling (A) Fluorescence images of different antibodies co- labeled with VALID, including parvalbumin (PV), glial fibrillary acidic protein (GFAP), tyrosine hydroxylase (TH), NeuN, ionized calcium binding adapter molecule 1 (Iba1), and microtubule-asso- ciated protein 2 (MAP2). (B and C) 3D reconstruction of the labeled NeuN- positive cellsandVALID-castedvascular structures, respectively. Merged images shown in (B) and (C). (D) Optical sections of reconstructed images shown in (C) at different imaging depths.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-GFAP primary antibody Proteintech Cat#16825-1-AP, RRID:AB_2109646 Goat anti-rabbit secondary antibody Alexa Fluor 488 Abcam Cat#ab150077, RRID:AB_2630356 Anti-CD31 Alexa Fluor 647 BioLegend Cat#102416, RRID:AB_493410 Anti-Tyrosine Hydroxylase Rabbit pAb Servicebio Cat#GB11181, RRID: AB_2921651 NeuN Polyclonal antibody Proteintech Cat#26975-1-AP, RRID:AB_2880708 Parvalbumin Polyclonal antibody Proteintech Cat#29312-1-AP, RRID: AB_2918280 CD31 Mouse Monoclonal antibody Proteintech Cat#66065-2-Ig, RRID: AB_2918476 MAP2 antibody Proteintech Cat# 17490-1-AP, RRID:AB_2137880 Iba1 antibody Abcam Cat# ab5076, RRID:AB_2224402 Chemicals, peptides, and recombinant proteins PBS Sigma-Aldrich Cat#P3813 Paraformaldehyde Sigma-Aldrich Cat#158127 Pork skin gelatin Sigma-Aldrich Cat#V900863 DiI Aladdin Cat#D131225 DiO Aladdin Cat#D131213 DiD Thermo Fisher Scientific Cat#D7757 MeOH Sinopharm Chemical Reagent Co. Ltd. Cat#10014118 EtOH Sinopharm Chemical Reagent Co. Ltd. Cat#10009218 Tert-butanol Sigma-Aldrich Cat#360538 Tetrahydrofuran Sigma-Aldrich Cat#186562 DBE Sigma-Aldrich Cat#108014 MXDA Tokyo Chemical Industry Cat#D0127 Sorbitol Sigma-Aldrich Cat#85529 Triton X-100 Sigma-Aldrich Cat#T8787 L. esculentum (Tomato) lectin Vector Laboratories Cat#DL1178 Sodium azide Sigma-Aldrich Cat#S2002 DAPI Thermo Fisher Scientific Cat# 62248 Wheat germ agglutinin, Alexa Fluor TM 647 conjugate Thermo Fisher Scientific Cat# W32466 Evans Blue Sigma-Aldrich Cat# E2129 Experimental models: Organisms/strains C57BL/6J Jackson Laboratories Cat# JAX:000664, RRID:IMSR_JAX:000664 Software and algorithms Matlab Mathworks https://www.mathworks.com/ products/matlab.html Imaris Bitplane https://www.bitplane.com/ imaris/imaris Fiji 55 https://imagej.nih.gov/ij/ Other Conical tubes (50 mL) Corning Cat#430828 Conical tubes (15 mL) Corning Cat#430790 (Continued on next page) Cell Reports Methods 3, 100407, February 27, 2023 e1

Techniques: Immunolabeling, Fluorescence, Labeling, Binding Assay, Imaging

Journal: Cell & Bioscience

Article Title: Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

doi: 10.1186/s13578-023-00954-3

Figure Lengend Snippet: EF stimulation increased neuronal differentiation of NSCs. A . Neurospheres and monolayer culture of NSCs for EF stimulation ( B ). C . The 7-day EF stimulation increased MAP2 (neuronal differentiation) and decreased GFAP (glial differentiation) protein expression in NSCs. D . EF stimulation for 7, 14 and 21 days increased neuronal, and reduced glial differentiation of NSCs. E , F . Quantification of MAP2 + and GFAP + NSCs counts with/without 7, 14 and 21-day EF stimulation. G . EF stimulation improved neurites process (βIII-tubulin + process) and synapse generation (Synapsin +). H Quantification of neurites process with/without 7, 14, 21-day EF stimulation. Scale bars: 20 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered as significantly different between EF and NoEF groups

Article Snippet: After blocking the non-specific proteins with 3% BSA-PB solution, the cells were then incubated with the primary antibodies: MAP2 (1:200, #4542S, Cell Signalling Technology, MA, USA), GFAP (1:200, #3670S, Cell Signalling Technology, MA, USA), βIII-tubulin (1:200, #5568, Cell Signalling Technology, MA, USA), Synapsin (1:200, #MA5-31919, ThermoFisher, USA), Synaptophys (1:200, #ab8049, Abcam, UK), Ki67 (1:1000, 9129S, Cell Signaling Technology, MA, USA) and Alexfluor 594/488-conjugated secondary antibodies (1:1000, Invitrogen, UK).

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

doi: 10.1186/s13578-023-00954-3

Figure Lengend Snippet: PI3Kγ deficiency abolished the PI3K/Akt/GSK-3β/β-catenin activation and neuronal differentiation of NSCs induced by EF stimulation. A – D . EF stimulation failed to increase the Akt and GSK-3β (Ser9) phosphorylation and β-catenin nuclear expression in PI3Kγ −/− NSCs. E – F . EF stimulation showed no effect on expression of MAP2 or GFAP in PI3Kγ −/− NSCs. G - J . EF stimulation failed to increase the Akt and GSK-3β (Ser9) phosphorylation and β-catenin nuclear expression in PI3Kγ KD/KD NSCs. K , L . EF stimulation showed no effect on expression of MAP2 or GFAP in PI3Kγ KD/KD NSCs. M . Schematic diagram: PI3Kγ was required in EF stimulation induced PI3K/Akt/GSK-3β/β-catenin pathway activation and neuronal differentiation in NSCs. Scale bars: 20 μm. * P < 0.05 was considered as significantly different between EF and NoEF groups

Article Snippet: After blocking the non-specific proteins with 3% BSA-PB solution, the cells were then incubated with the primary antibodies: MAP2 (1:200, #4542S, Cell Signalling Technology, MA, USA), GFAP (1:200, #3670S, Cell Signalling Technology, MA, USA), βIII-tubulin (1:200, #5568, Cell Signalling Technology, MA, USA), Synapsin (1:200, #MA5-31919, ThermoFisher, USA), Synaptophys (1:200, #ab8049, Abcam, UK), Ki67 (1:1000, 9129S, Cell Signaling Technology, MA, USA) and Alexfluor 594/488-conjugated secondary antibodies (1:1000, Invitrogen, UK).

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Cell & Bioscience

Article Title: Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

doi: 10.1186/s13578-023-00954-3

Figure Lengend Snippet: Knock down of β-catenin abolished the neuronal differentiation of NSCs induced by EF stimulation. A , B The β-catenin expression in NSCs was knocked down with siCTNNB1 transfection. Scale bar: 10 μm. C , D EF stimulation increased NeuroD1 in WT NSCs. E , F EF stimulation failed to increase (or even in reversing trend) NeuroD1 expression in siCTNNB1 transfected NSCs. G , H . EF stimulation showed no effect on expression of MAP2 or GFAP in siCTNNB1 transfected NSCs. Scale bar: 20 μm. I . Schematic diagram: β-catenin is required in EF stimulation induced the NeuroD1 and neuronal differentiation of NSCs. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered as significantly different between EF and NoEF groups

Article Snippet: After blocking the non-specific proteins with 3% BSA-PB solution, the cells were then incubated with the primary antibodies: MAP2 (1:200, #4542S, Cell Signalling Technology, MA, USA), GFAP (1:200, #3670S, Cell Signalling Technology, MA, USA), βIII-tubulin (1:200, #5568, Cell Signalling Technology, MA, USA), Synapsin (1:200, #MA5-31919, ThermoFisher, USA), Synaptophys (1:200, #ab8049, Abcam, UK), Ki67 (1:1000, 9129S, Cell Signaling Technology, MA, USA) and Alexfluor 594/488-conjugated secondary antibodies (1:1000, Invitrogen, UK).

Techniques: Knockdown, Expressing, Transfection

Journal: Cell & Bioscience

Article Title: Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

doi: 10.1186/s13578-023-00954-3

Figure Lengend Snippet: EF pre-stimulated NSCs transplantation improved neurogenesis and motor function recovery of spinal cord injury. A – C . The mRNA expressions of NES (nestin, NSCs marker), MAP2 (neuronal differentiation) and GFAP (glial differentiation) from the spinal cord of the SCI mice with NSCs transplantation. D BBB assessment (average score of right and left hind limbs) for hind limb motor function recovery of the SCI mice with NSCs transplantation. The mice with EF pre-stimulated WT NSCs transplantation demonstrated better motor function repairmen than NoEF WT NSCs. Whilst, with the PI3Kγ −/− NSCs, the effect of EF pre-stimulation was abolished. A group with PBS injection was used as a negative control. E Exemplar photos of SCI mice with EF and NoEF pre-stimulated NSCs transplantation, 28 DAT. The SCI mouse receiving EF pre-stimulated NSCs transplantation showed occasional weight-supported dorsal stepping on right hind limb (left). While the SCI mouse receiving NoEF treated NSCs transplantation showed slight movement of both hind limbs, without any weight-support. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered as significantly different between EF and NoEF groups

Article Snippet: After blocking the non-specific proteins with 3% BSA-PB solution, the cells were then incubated with the primary antibodies: MAP2 (1:200, #4542S, Cell Signalling Technology, MA, USA), GFAP (1:200, #3670S, Cell Signalling Technology, MA, USA), βIII-tubulin (1:200, #5568, Cell Signalling Technology, MA, USA), Synapsin (1:200, #MA5-31919, ThermoFisher, USA), Synaptophys (1:200, #ab8049, Abcam, UK), Ki67 (1:1000, 9129S, Cell Signaling Technology, MA, USA) and Alexfluor 594/488-conjugated secondary antibodies (1:1000, Invitrogen, UK).

Techniques: Transplantation Assay, Marker, Injection, Negative Control

Journal: Cell & Bioscience

Article Title: Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

doi: 10.1186/s13578-023-00954-3

Figure Lengend Snippet: List of the primers used in this study

Article Snippet: After blocking the non-specific proteins with 3% BSA-PB solution, the cells were then incubated with the primary antibodies: MAP2 (1:200, #4542S, Cell Signalling Technology, MA, USA), GFAP (1:200, #3670S, Cell Signalling Technology, MA, USA), βIII-tubulin (1:200, #5568, Cell Signalling Technology, MA, USA), Synapsin (1:200, #MA5-31919, ThermoFisher, USA), Synaptophys (1:200, #ab8049, Abcam, UK), Ki67 (1:1000, 9129S, Cell Signaling Technology, MA, USA) and Alexfluor 594/488-conjugated secondary antibodies (1:1000, Invitrogen, UK).

Techniques: Sequencing