Journal: Cell

Article Title: Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels

doi: 10.1016/j.cell.2018.06.007

Figure Lengend Snippet: Functional Validation of Genetic Reagents and Test of Interaction between Robo and Notch Signaling, Related to , , and (A and B) Validation of dnRobo and myrRobo as dominant-negative and constitutively active for Robo signaling, respectively. In (A), growth cone collapse assay of growing axons from explants of embryonic mouse retinas, electroporated to express Gfp or dnRobo and exposed to recombinant Slit protein or vehicle solution. Failure of response to Slit upon dnRobo-overexpression demonstrates its dominant-negative effect (n = 44-58 growth cones per group, 3 independent experiments). In (B), branching assay of growing axons from single neurons of embryonic rat dorsal root ganglion, overexpressing Gfp alone or with myrRobo constructs as indicated. Exuberant axonal branching typically elicited by Slit-Robo signaling occurs in myrRobo-expressing neurons in the absence of Slit, demonstrating constitutive activation of Robo signaling (n = 5-10 neurons per group). (C–F) Validation of crispr constructs for disruption of Dll1 in mouse and human. (C) Top, sequence of the gRNA targeting mouse Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the mDll1 coding sequence (gray bar). Bottom, validation of Crispr-mediated editing of the mDll1 locus upon electroporation with gDll1 plus Cas9, but not with Cas9 alone. Different lanes correspond to independent electroporated embryos. M, molecular weight marker. Arrow indicates 1,025 bp amplicon, arrowheads indicate the products of PCR amplicon digestion by Syrveyor Nuclease (656 + 368 bp), absent in the Cas9-alone lanes. (D) Left, sequence of the gRNA targeting human Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the hDll1 coding sequence (gray bar). Right, chromograms for genome sequence validation of Crispr-mediated editing of the hDll1 locus upon electroporation of cerebral organoid with gDll1 plus Cas9. A 270bp fragment was inserted at position 54 of the coding sequence, introducing a STOP codon in position 76. (E and F) Effect of electroporating crDll1 in NCx (green cells) on the abundance of Dll1 protein (red). Details are examples VZ cells loosing Dll1 protein (arrowheads) from the cell surface upon crDll1 (n = 3 embryos per group). (G–K) Antibody stain for GFP and Robo1 or Dll1 in NCx at E13.5 upon electroporation of the indicated plasmid combinations at E12.5, and quantifications (paired t test). Arrowheads indicate area of increased Robo (n = 3 embryos per group). Values are mean + SEM; paired or independent samples t tests; ∗ p < 0.05; ns, not significant. Scale bars: 30 μm (E), 50 μm (G and H).

Article Snippet: After 24h of incubation, recombinant mouse Slit protein (R&D Systems) was added to the medium (250ng/μl) and the explants were fixed 1h later with PFA 2% during 15 min.

Techniques: Functional Assay, Biomarker Discovery, Dominant Negative Mutation, Recombinant, Over Expression, Construct, Expressing, Activation Assay, CRISPR, Disruption, Sequencing, Electroporation, Molecular Weight, Marker, Amplification, Staining, Plasmid Preparation

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Transcriptional mechanism underpinning Slit3 secretion. a mRNA expression levels of Ets1, E47, FoxJ2 , and FoxA2 genes in MC3T3 cells cultured in osteoblast differentiation-inducing medium and treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, t -test). b , c Protein expression level of E47, FoxA2 in MC3T3 cells cultured in stimulated medium and treated with PTH or Veh (PBS) for 3 days ( n = 3, t -test). Representative images depicting E47 IF staining (green) in lumbar vertebral body and endplate sections ( d ), quantitative analysis of the number of E47 + cells in the vertebral body ( e ) and endplate ( f ) of aged mice treated with Veh or PTH ( n = 5, t -tset). Scale bar: 100 µm. Representative images showing FoxA2 IF staining (red) in lumbar vertebral body and endplate sections ( g ), quantitative analysis of the number of FoxA2 + cells in the vertebral body ( h ) and endplate ( i ) of aged mice treated with PTH or vehicle for 2 months ( n = 5, t -tset). Scale bar: 100 µm. j Relative fold enrichment of the E47 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). k Relative fold enrichment of the FoxA2 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Binding Assay

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Efficacy of PTH treatment is diminished in osteoblastic Slit3 knockout mice with spinal degeneration. Vertebral endplate bone structure analyses by micro-CT: bone volume per tissue volume (BV/TV) percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in Slit3 OCN −/− LSI mice with PTH (40 µg/kg/d) or Veh treatment for 2 months ( n = 7, t -test). Behavior evaluations included pressure tolerance of the lumbar spine assessed via the force threshold ( d ), total distance covered in spontaneous activity in 2 days ( e ), and latency of paw withdrawal post-thermal stimulation ( f ) for Slit3 OCN −/− LSI mice with PTH or Veh treatment for 2 months ( n = 7, t -test). g Western blot analysis of protein expression levels of β3 tubulin, CGRP, PGP9.5 relative to GAPDH in endplate tissue (L3-L5) in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months. ( n = 5). Representative images of PGP9.5 (red) and CGRP (green)-positive fibers ( h ) and quantitative analysis of fiber length in the lumbar vertebral body of Slit3 OCN −/− LSI mice with PTH or Veh treatment ( i , j ) ( n = 5, t -tset). Scale bar: 100 µm. k Western blot analysis of protein expression levels of CGRP in DRG tissue (L1–L2), normalized to GAPDH expression, in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5). Representative IF images showing CGRP-positive (green) neurons in DRG sections ( l ), followed by quantitative analysis of the mean IF intensity for CGRP ( m ) in the DRG of Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5, t -tset). Scale bar: 100 µm

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Knock-Out, Micro-CT, Activity Assay, Western Blot, Expressing

Journal: Current biology : CB

Article Title: FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

doi: 10.1016/j.cub.2014.01.042

Figure Lengend Snippet: Figure 2. Slit1 Enables Netrin-1 Attraction by Activating the Robo1 Receptor

Article Snippet: Recombinant Slit1 (5 nM; R&D Systems), FLRT3-ECD [15] (1 mg/ml), a mouse anti-DCC antibody (AF5 clone; Abcam), or control mouse IgG was (J and K) Robo1+/+ rTCAs grow rostrally as a compact bundle (J), whereas the (L) Quantification of the data represented in (J) and (K). rTCA dispersion at the v tailed Student’s t test. (M) Scheme representing the topographic guidance of rTCAs and iTCAs in t Slit1 and Netrin-1 [21].

Techniques:

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: Effect of SLIT1 supplementation on axonal growth of hESC- and hiPSC-derived RGCs. ( a ) Axonal growth of hESC- and hiPSC-derived RGCs is observed by immunostaining of NFL. In the control, hESC- and hiPSC-derived RGCs axons grow radially and straight (left panels). In contrast, the growth pattern of axons after SLIT1 supplementation is complex. Axonal growth of hESC- and hiPSC-derived RGCs is not inhibited by addition of 0.2 and 1.0 µg of SLIT1 supplementation from D27−30 (centre and right panels, respectively). However, axonal paths are disturbed (centre and right panels). The assessment at D27 is performed in RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. ( b ) Real-time PCR analysis of mRNA expression of axonal markers, TAU , NFL , and TUJ1 . Expression levels of axonal markers are not influenced by SLIT1 supplementation at any of the concentrations tested. Scale bar, 100 μm. Error bars indicate ± SD. Each column shows an average value for the studied samples. The sample size for all mRNA data is five (n = 5). NS, not significant.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Immunostaining, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: Effect of locally sustained release of SLIT1 (5μg/ml) from hydrogels placed in front of the attached OVs. Locally sustained release of SLIT1 using hydrogel does not affect axonal growth of hESC- and hiPSC-derived RGCs (( a ) and ( d ), respectively, magnification 40x). Only a subset of axons is repelled by SLIT1 release (( b ) and ( e ), magnification 100x: squares in ( a ) and ( d ), respectively), while other axons remain unaffected. Filopodia of hESC- and hiPSC-derived RGCs (( c ) and ( f ), respectively) have collapsed in response to SLIT1 release, as evident from phalloidin and GAP43 staining. The assessment from D27 is performed under RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. Each experiment was repeated at least five times. Squares in ( a ) and ( d ) correspond to ( b ) and ( e ), respectively. Scale bars in ( a ) and ( d ), 100 μm. Scale bars in ( b ) and ( e ), 40 μm. Scale bars in ( c ) and ( f ), 10 μm.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Staining

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: The effect of focally sustained release of SLIT1 on pathfinding of axons derived from hESC- and hiPSC-derived RGCs. Phase-contrast images showing SLIT1-releasing beads (blue-coloured) placed next to the bottom of attached OV, and corresponding immunohistochemistry for the axons of the RGCs by NFL staining. ( a,c ) The paths of the axons of hESC- and hiPSCs- derived RGCs (( a ) and ( c ), respectively) stained by NFL radiate straight from the attached OV, even though the bead is located close by. ( b , d ) Compared with the control, the axons of hESC- and hiPSC-derived RGCs (( b ) and ( d ), respectively) are repelled by SLIT1 release from the bead, and axonal paths are not straight, but winding, and avoid the beads. The assessment from D27 is performed in RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. Each experiment was repeated at least five times. Scale bars, 100 μm.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Immunohistochemistry, Staining, Control

Journal: Scientific Reports

Article Title: Transcriptome and methylome analysis reveals three cellular origins of pituitary tumors

doi: 10.1038/s41598-020-76555-8

Figure Lengend Snippet: ( A ) PCA of pituitary adenoma (PA) transcriptome showing three distinct clusters: POU1F1-driven GH-, TSH- and PRL-adenomas; NR5A1-driven gonadotropinomas and null cell adenomas; and TBX19-driven clinically evident ACTH adenomas. Silent ACTH PA grouped separately sharing features with both, TBX19-dependent and NR5A1-dependent adenomas. ( B ) Heatmap of the differentially expressed genes. In the “Y” axis tumor samples are grouped according to the World Health Organization (WHO) 2017 classification as gonadotrope cell adenomas, null-cell adenomas, clinically evident ACTH adenomas, clinically silent ACTH adenomas, somatotrope adenomas, prolactinomas and TSH adenomas; tumors are also classified in the figure according to clinical features such as size, invasion, recurrence and aggressiveness. The “X” axis represents the differentially expressed genes hierarchical cluster. ( C ) CACNA2D4 is upregulated in NR5A1-driven tumors; ( D ) EPHA4 is upregulated in TBX19-driven tumors; ( E ) SLIT1 is upregulated in POU1F1-driven tumors. Image was created using Partek Genomics Suite 7.19v ( https://www.partek.com/partek-genomics-suite/ ).

Article Snippet: For RT-qPCR of SLIT1 (Hs00171488_m1), EPHA4 (Hs00953178_m1) and CACNA2D4 (Hs00297782_m1) all reagents were purchased from Applied Biosystems (CA, USA), and conditions were as follows: 10 μl of Taqman Universal Master Mix II, 1 μl of each Taqman probe, 200 ng of cDNA in a 20 μl final volume, according to manufacturer’s recommendation.

Techniques:

Journal: Scientific Reports

Article Title: Transcriptome and methylome analysis reveals three cellular origins of pituitary tumors

doi: 10.1038/s41598-020-76555-8

Figure Lengend Snippet: ( A – C ) Dot blots of representative up regulated genes. EPHA4 in TBX19-derived tumors , CACNA2D4 in NR5A1-derived tumors and SLIT1 in POU1F1-derived tumors. ( D – F ) Violin plots showing validation of gen up-regulation in each tumor group by RT-qPCR. Image was created using Partek Genomics Suite 7.19v ( https://www.partek.com/partek-genomics-suite/ ).

Article Snippet: For RT-qPCR of SLIT1 (Hs00171488_m1), EPHA4 (Hs00953178_m1) and CACNA2D4 (Hs00297782_m1) all reagents were purchased from Applied Biosystems (CA, USA), and conditions were as follows: 10 μl of Taqman Universal Master Mix II, 1 μl of each Taqman probe, 200 ng of cDNA in a 20 μl final volume, according to manufacturer’s recommendation.

Techniques: Derivative Assay, Biomarker Discovery, Quantitative RT-PCR

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Dynamic changes in Slit1 expression in DRG after sciatic nerve injury. The expression of Slit1 in DRG was detected by immunofluorescence histochemistry and western blotting at different time points after SNC. (A–E) Contralateral DRG sections. (F–J) Ipsilateral DRG sections. The white arrows indicate strongly Slit1-positive neurons, and the green arrow indicates weakly Slit1-positive neurons. Scale bar = 100 μm. (K) At different time points, the numbers of strongly Slit1-positive DRG neurons on both sides were compared, and one-way repeated measures ANOVA and paired t -tests were used for statistical analysis. “ ∗ ” in comparison with the control side, the difference is statistically significant at P < 0.05; “#” in comparisons of the groups at 1, 3 or 7 days after injury with the group at 14 days after injury, the difference is statistically at P < 0.01. (L) Detection of DRG Slit1 protein expression at different time points by western blotting ( N = 3). The level of β-actin was detected as loading control. (M) At different time points, the relative gray values of DRG Slit1 expression on the two sides were compared. N = 3, and one-way repeated measures ANOVA and paired t -tests were used for statistical analysis, “ ∗ ” compared to the contralateral side, P < 0.01. CON: contralateral DRG, IPS: ipsilateral (crush side) DRG.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Western Blot, Comparison, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Slit1 expression in SGCs of injured DRG. Slit1 expression in the SGCs of injured DRG was detected by immunofluorescence histochemistry. (A) Slit1 (red) immunofluorescence staining of contralateral DRG sections randomly selected from the different groups. (B–F) Slit1 immunofluorescence staining of ipsilateral DRG at different time points after operation. The arrow indicates Slit1-positive SGCs. (G) GS (green) immunofluorescence staining of DRG sections on the 7th day after sciatic nerve crush. (H) Merged images from panels (D,E) . (I) Summary histograms depicting the changes in the Slit1 RFI in SGCs (one-way repeated measures ANOVA, ∗ P < 0.01); RFI, relative fluorescence intensity. (J) Changes in the number of strongly Slit1-positive DRG neurons and the RFI of Slit1 expression in SGCs at different time points after injury are shown. Scale bar = 100 μm.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Effects of DRG-injured neuronal soma on Slit1 expression in SGCs. The Slit1 and ATF3 double immunofluorescence labeling method and the Slit1 and FG retrograde labeling method were used to show the expression of Slit1 in SGCs around normal and injured neurons. (A–C) Double immunofluorescence labeling of Slit1 and ATF3 was carried out on DRG sections from 7 days after sciatic nerve crush. (A) Slit1 (red) immunofluorescence staining. (B) ATF3 (green) immunofluorescence staining. (C) Merged images of panels (A,B) . The thick arrow indicates ATF3-positive neurons, and the thin arrow indicates ATF3-negative neurons, scale bar = 25 μm. (D–F) Slit1 immunofluorescence staining and FG retrograde labeling. (D) DRG Slit1 immunofluorescence on the 7th day after the operation. (E) FG (white)-traced positive neurons. (F) Merged images of panels (D,E) . Scale bar = 200 μm. The large framed image is an enlargement of the small frame, and thin arrows indicate FG-negative neurons in panels (F) . (G) Summary histograms depict the changes in the RFI of Slit1 in SGCs surrounding ATF3-positive and negative neurons determined with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity. (H) Summary histograms depict the changes in the RFI of Slit1 expression in SGCs surrounding FG-negative and FG-positive neurons determined with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Expression of Slit1 in SGCs directly in contact with the neuronal soma in vitro (A–C) Primary cultured DRG cells were double labeled with Slit1 and MAP2 immunochemistry, nuclear staining by DAPI. (A) Slit1 (red) and DAPI (blue) merged image. The box 1 shows SGCs in contact with neuron bodies, Panel (A1) is its enlarged image, with arrows indicating SGCs in contact with neuronal soma (white asterisks); and the box 2 shows SGCs located far from neuron bodies as showed in its enlarged image (A2) . (B) MAP2 (green) image. (C) Slit1 (red), MAP2 (green) and DAPI (blue) merged image. (D) Slit1 and GS double immunostaining were performed on cultured DRG cells, nuclear staining by DAPI. The box 1 shows SGCs in contact with Slit1-positive neuronal soma (white asterisks). The box 2 shows SGCs located far from neuron bodies. Panels (D1,D2) lines of pictures were local enlarged images of the box 1 and box 2 showed in panel (D) , respectively. The white asterisk indicates the neuronal soma. Slit1 (red), MAP2 (green) and DAPI (blue) were showed in panels (D,D1,D2) . (E) Summary histograms depict the changes in Slit1 expression in two different populations of SGCs, ∗ shows the expression of Slit1 in SGCs of population 1 compared with that in SGCs of population 2 analyzed with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity. Scale bar = 50 μm was showed in panels (A–C) ; Scale bar = 15 μm was showed in panel (D) .

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, In Vitro, Cell Culture, Labeling, Staining, Double Immunostaining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Slit1 expression in SGCs decreased after inhibition of the P2X7 receptor with BBG. After right sciatic nerve crush, BBG (P2X7 receptor inhibitor) was injected intraperitoneally. The expression of Slit1 on the 7th day DRG was detected by double immunofluorescence staining and western blotting. (A–D) Slit1 immunofluorescence staining (red). (E–H) Merged images of Slit1 (red) and GS (green) immunofluorescence staining. Scale bar = 200 μm. (I) the level of Slit1 protein was detected by western blotting ( N = 3), the level of β-actin was detected as loading control. (J) The level of Slit1 before and after BBG treatment on the 7th day post-SNC DRG was analyzed with the paired t -test. “ ∗ ” compared with the control side, the level of Slit1 in IPS side was upregulated, P < 0.001; “#” compared with the control side and the ipsilateral side before BBG treatment respectively, the level of Slit1 in “IPS + BBG” was downregulated, P < 0.001; The change was not significant in “CON + BBG”, P > 0.05. “ ∗∗ ” compared with “CON + BBG” group, the level of Slit1 in “IPS + BBG” was much lower than that in “CON + BBG” group, P < 0.001. CON: contralateral DRG, IPS: ipsilateral DRG.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Inhibition, Injection, Double Immunofluorescence Staining, Western Blot, Immunofluorescence, Staining, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Schematic diagram of the role of Slit1 in the communication between neurons and SGCs. After peripheral nerve injury, neurons activate perineuronal SGCs, leading to upregulation of Slit1 expression through the VNUT-ATP-P2X7R pathway, and SGCs then promote the regeneration of injured neurons through the Slit1-Robo2-srGAP3 pathway ( ; ).

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Primary and secondary antibodies used in this study.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Control