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: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Quantitative RT-qPCR primer sequences

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Antibodies

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Western Blot, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Slit and Robo genes are expressed in the Leydig cells of the mouse testis. a RT-qPCR analyses of brain, testicular and purified Leydig cell Slit and Robo mRNA levels (n = 4–6 per group). The relative levels are represented as delta-Ct versus Actb . Data are means ± sem. b SLIT1, -2 and -3 and ROBO1 immunohistochemistry analyses of 8 week-old wild-type mouse testis

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Quantitative RT-PCR, Purification, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases steroidogenesis in Leydig cells in vitro. Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in MA10 cells treated a for 4 h with vehicle or 1, 5 and 10 µg/ml exogenous SLIT2; b for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. c Representative graph of progesterone concentrations (corrected to RNA input) measured in the spent culture media of MA10 cells treated for 8 h with vehicle or 10 µg/ml SLIT2. d Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR and e representative graph of testosterone concentrations (corrected to RNA input) measured in the spent culture media of mouse primary Leydig cell cultures treated for 8 h with vehicle or 10 µg/ml SLIT2. Experiments were performed three times in triplicate. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: In Vitro, Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases CREB phosphorylation. a Quantification of total and phosphorylated CREB, AKT and mTOR protein levels normalized to ACTB in MA10 cells treated for 1 h with vehicle or 10 µg/ml SLIT2. Quantification of total and phosphorylated AKT and CREB protein levels normalized to GAPDH, and expression of Star determined by RT-qPCR, in MA10 cells treated with vehicle or b 100 nM wortmannin, or c 20 µg/ml SC79, for 1 h (protein) or 4 h (mRNA). n = 3–9 samples per group. Expression of Star was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 alters Leydig cell responsiveness to LH by decreasing Lhcgr expression. a Expression of Lhcgr determined by RT-qPCR in MA10 cells treated for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. b Expression of Star , Cyp11a1 , Cyp17a1 and Hsd3b1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 4 h. c Quantification of total and phospho-CREB protein levels normalized to GAPDH in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 30 min. d Expression of Star and Cyp11a1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 10 µM forskolin for 4 h. n = 3–9 samples per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001; NS not significant

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Increased testicular steroidogenesis in Robo1- null mice. a Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in primary Leydig cells isolated from Robo1 +/+ vs Robo1 Robo1 −/− mice treated for 8 h with vehicle or 10 µg/ml SLIT2. n = 4 samples per group. b Expression of Robo2 determined by RT-qPCR in cultured Leydig cells isolated from Robo1 +/+ and Robo 1 −/− mice. n = 4 samples per group. c Expression of Star , Cyp11a1 , Cyp17a1 , Hsd3b1 and Lhcgr determined by RT-qPCR in testes from four month-old Robo1 +/+ and Robo1 −/− mice. n = 5–7 per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . d Intra-testicular testosterone concentrations corrected to testis weight measured in two- and four month-old Robo1 +/+ and Robo1 −/− mice, and serum LH levels measured in four month-old mice. n = 5–10 per group. Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture

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 (R&D Systems) and SEMA3A (R&D Systems) diluted in DPBS were used at a concentration of 200 ng/ml, and recombinant human SLIT1 (R&D Systems) diluted in DPBS was used at a concentration of 5 μg/ml.

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 (R&D Systems) and SEMA3A (R&D Systems) diluted in DPBS were used at a concentration of 200 ng/ml, and recombinant human SLIT1 (R&D Systems) diluted in DPBS was used at a concentration of 5 μg/ml.

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 (R&D Systems) and SEMA3A (R&D Systems) diluted in DPBS were used at a concentration of 200 ng/ml, and recombinant human SLIT1 (R&D Systems) diluted in DPBS was used at a concentration of 5 μg/ml.

Techniques: Derivative Assay, Immunohistochemistry, Staining, Control

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:

Article Title: Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals

doi: 10.1016/j.mcn.2010.11.003

Figure Lengend Snippet: TH+ axons project adjacent to Slit+ tissue, and express Robo1 and 2. A. Schematic representation of the initial mdDA projections through the diencephalon. The nucleus of the mdDA neurons is positioned along the cephalic flexure and extends across the forebrain/midbrain boundary. These TH+ axons project anteriorly in a distinct ventrolateral position through the thalamus. Adjacent to the MFB tract, the A13 cell population serves as a landmark. Black lines marked E-F and G-H indicate section planes in those panels. B. Wild-type whole mount embryo in the CD-1 background immunolabeled with TH. The white arrow represents the normal trajectory of mdDA axons through the thalamus. The tract is labeled MFB. C. Slit1 mRNA expression (purple) is localized around the cephalic flexure, with robust signal at the ventral midline (arrowhead). Fainter graded expression domains are visible in dorsal midbrain, and dorsal and ventral thalamus. D. Slit2 expression is also seen along the ventral midline, overlapping with the expression of Slit1. Very faint expression is also found in the hypothalamus and lateral thalamus. E. Robo1 (red) and TH (green) antibody labeling of a sagittal section of an E12.5 embryo. Robo1 is expressed in an extensive set of longitudinal bundles (E) which transit the mdDA nucleus. F-H. High magnification views of a different section. Some TH cell bodies are Robo1+ (yellow arrowheads), while other TH+ cell bodies do not show detectable Robo1 labeling (green arrowheads). The dotted lines trace a few examples of TH+ axons, of which some are closely associated with Robo1+ bundles (yellow), while other TH+ axons are not (green). I. Robo2 antibody labeling of a sagittal section of an E12.5 embryo. While Robo2 label can be seen in longitudinal fibers in dorsal, anterior, and posterior regions, little to no Robo2 label can be seen in the area of the mdDA or the MFB. J, K. Adjacent tissue sections showing anti-β-galactosidase immunolabeling representing expression from a Robo2lacZ allele (J), in comparison to TH in the MFB (K). Both markers label MFB fibers (yellow arrows). Abbreviations: cf, cephalic flexure; cv, cerebral vesicle; DT, dorsal thalamus; Hyp, hypothalamus; M, midbrain; md, mesodiencephalon; MFB, medial forebrain bundle; op, optic stalk; VT, ventral thalamus. Scale Bars: B and E, 400 μm; (applies to C, D, I); F, 50 μm; (applies to G, H); K, 100 μm (applies to J).

Article Snippet: Slit1 -/- ; Slit2 + is a pool of control embryos (n=8) derived from the breeding stock of Slit1 homozygotes, with or without a Slit2 - allele.

Techniques: Immunolabeling, Labeling, Expressing, Antibody Labeling, Comparison

Journal:

Article Title: Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals

doi: 10.1016/j.mcn.2010.11.003

Figure Lengend Snippet: Slits organize mdDA projections through the diencephalon, with a dominant role for Slit2. Whole-mount TH immunolabeling and schematics of E12.5 (A-I) and E13.5 (J-M) embryos at low (A,D,G,J) and high (B,E,H,K) magnification. A-C. TH+ axons in Slit1-/-;Slit2+/- double mutants, with projections identical to wildtype controls (compare to Fig 1B). Note that TH+ axons project in an organized and relatively narrow ascending trajectory through the diencephalon toward the telencephalon. Axons avoid the ventral hypothalamus, and project ventral to the A13 landmark. D-F. Slit1-/-; Slit2-/- double homozygotes form a wider tract, with many axons growing within ventral midline tissue near the cephalic flexure (E). In addition, axons have shorter projections with few in the proper direction, and some wandering dorsally. G-I. Slit1+/+; Slit2-/- single mutant embryos are similar to Slit1-/-; Slit2-/- double mutants, with projections looping into the ventral midline (*), extending over a wider dorsoventral breadth, and of shorter length than in control embryos. J-L. By E13.5 the ventral looping into the midline is still prevalent and mdDA neurons continue to project over a wider dorsoventral breadth, invading ventral midline tissue. K. Within the anterior diencephalon, mdDA axons deviate from the tract to project toward the optic stalk (arrow). Axons in the tract converge abnormally (arrowhead). M. Quantitation of the width of the mdDA tract, measured at the point indicated in the schematic summary of each mutant phenotype. Tract width is indicated by arbitrary units consistent between the genotype pools, +/- SEM. Slit1-/-; Slit2+ is a pool of control embryos (n=8) derived from the breeding stock of Slit1 homozygotes, with or without a Slit2- allele. Slit1+; Slit2-/- are a similar pool of embryos with at least one Slit1+ allele (n=6), compared to Slit1-/-; Slit2-/- double mutants (n=7). By ANOVA, the three pools were significantly different. cf, cephalic flexure; DT, dorsal thalamus; Hyp, hypothalamus; M, midbrain; VT, ventral thalamus. Scale bars, in A and B represent 400 μm for low and high magnification, respectively.

Article Snippet: Slit1 -/- ; Slit2 + is a pool of control embryos (n=8) derived from the breeding stock of Slit1 homozygotes, with or without a Slit2 - allele.

Techniques: Immunolabeling, Mutagenesis, Control, Quantitation Assay, Derivative Assay

Journal:

Article Title: Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals

doi: 10.1016/j.mcn.2010.11.003

Figure Lengend Snippet: Robo1 and 2 cooperate to guide mdDA axons through the diencephalon. Whole-mount TH immunolabeling of E12.5 (A-F) and E13.5 (G-J) embryos shows Robo1/2 heterozygous (A-B) and double mutant (D-E, G-K) phenotypes at low (A,D,G) and high (B,E, I-K) magnification. A-C. Robo1+/-; Robo2+/- embryos show a tract identical to wildtype controls. D-F. Robo1-/-; Robo2-/-double mutants are have axons growing toward and entering the ventral midline near the cephalic flexure, dorsoventral broadening of the tract, and shorter projections with few in the proper direction, identical to the Slit1-/-; Slit2-/- double homozygous phenotype. Additionally, Robo1-/-; Robo2-/- double mutants exhibit extensive dorsal wandering with axons fasciculating along aberrant trajectories (* in D). G-K. E13.5 Robo1-/-; Robo2-/- double homozygotes have worsening errors, including dorsal wandering (J) and an anterior split in the tract, with some axons diverging anteriorly toward the optic stalk in the anterior forebrain (I). cf, cephalic flexure; DT, dorsal thalamus; Hyp, hypothalamus; M, midbrain; op, optic stalk; VT, ventral thalamus. Scale bars represent 400 μm.

Article Snippet: Slit1 -/- ; Slit2 + is a pool of control embryos (n=8) derived from the breeding stock of Slit1 homozygotes, with or without a Slit2 - allele.

Techniques: Immunolabeling, Mutagenesis