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: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: NETRIN-1 expression was significantly reduced, whereas SLIT3 expression was markedly elevated in the corneal epithelium of Aqp5 −/− mice compared to Aqp5 +/+ mice. ( A ) Heatmap illustrates differentially expressed neurotrophic and axon guidance factors in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( B ) Volcano plot analysis of differentially expressed neurotrophic and axon guidance factors between the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice, with upregulated genes highlighted in red and downregulated genes in blue . ( C ) Quantitative RT-PCR analysis of Ntn1 and Slit3 expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice ( n = 3). ( D ) Representative immunofluorescence images of corneas stained with NETRIN-1 and SLIT3 ( green ) and DAPI ( blue ). Scale bars : 20 µm. ( E ) Regional quantification of positive immunofluorescence staining from images in ( D ) ( n = 3). ( F ) Western blot analysis of NETRIN-1, SLIT3, and ROBO4 protein expression in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( G ) Statistical evaluation of Western blot data ( n = 6). ( H ) Immunofluorescence staining of primary corneal epithelial cells isolated from Aqp5 +/+ and Aqp5 −/− mice, labeled with DAPI ( blue ), NETRIN-1, and SLIT3 ( green ). Scale bars : 25 µm. ( I ) Regional quantification of positive immunofluorescence staining from images in ( H ) ( n = 6). ( J ) The protein concentration of NETRIN-1 and SLIT3 in primary corneal epithelial cell supernatant was detected through ELISA ( n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies against βIII-tubulin (#AB15708A4; Sigma-Aldrich, St. Louis, MO, USA), NETRIN-1 (#A16236; ABclonal), or SLIT3 (#DF9909; Affinity).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Isolation, Labeling, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: NETRIN-1 and SLIT3 affected neuronal axon growth. ( A ) Axonal growth status of Aqp5 +/+ and Aqp5 −/− TG neurons following treatment with recombinant mouse NETRIN-1 (rmNETRIN-1) and recombinant mouse SLIT3 (rmSLIT3). Scale bars : 50 µm. ( B ) Statistical analysis of the maximum branch lengths of TG neurons ( n = 6). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies against βIII-tubulin (#AB15708A4; Sigma-Aldrich, St. Louis, MO, USA), NETRIN-1 (#A16236; ABclonal), or SLIT3 (#DF9909; Affinity).

Techniques: Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: Effects of corneal epithelial TEs and NAbs on axon growth in TG neurons. ( A ) Left : Axon growth was assessed following the addition of Aqp5 −/− and Aqp5 +/+ corneal epithelial TEs to Aqp5 +/+ TG neurons. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( A , i ) ( n = 6). ( B ) Left : Axon growth was assessed following the addition of Aqp5 −/− and Aqp5 +/+ corneal epithelial TEs to Aqp5 −/− TG neurons. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( B , i ) ( n = 6). ( C ) Left : Axon growth following the simultaneous addition of Aqp5 −/− TEs or SLIT3 NAbs. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons presented in ( C , i ) ( n = 6). ( D ) Left : Axon growth following the simultaneous addition of Aqp5 +/+ TEs or NETRIN-1 NAbs. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( D , i ) ( n = 6). Results of statistical analyses are expressed as mean ± SD. *** P < 0.001.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies against βIII-tubulin (#AB15708A4; Sigma-Aldrich, St. Louis, MO, USA), NETRIN-1 (#A16236; ABclonal), or SLIT3 (#DF9909; Affinity).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: The transcription factor JUN-D bound to and regulated the expression of NETRIN-1 and SLIT3 in corneal epithelial cells. ( A ) Heatmap analysis of Ntn1 and Slit3 cotranscription factor expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( B ) Western blot analysis of JUN-D protein expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( C ) Quantitative statistical analysis of Western blot data ( n = 3). ( D ) ChIP-PCR analysis revealed significant enrichment of JUN-D in the promoter regions of Ntn1 and Slit3 . ( E ) Quantitative RT-PCR analysis was conducted to evaluate the expression levels of Ntn1 and Slit3 in corneal epithelial cells isolated from Aqp5 +/+ mice following 24-hour treatment with SR11302 ( n = 3). ( F ) Western blot analysis of NETRIN-1 and SLIT3 protein expression in the corneal epithelial cells of Aqp5 +/+ mice treated with SR11302. ( G ) Quantitative statistical evaluation of Western blot data ( n = 6). Statistical results are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies against βIII-tubulin (#AB15708A4; Sigma-Aldrich, St. Louis, MO, USA), NETRIN-1 (#A16236; ABclonal), or SLIT3 (#DF9909; Affinity).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Isolation, Standard Deviation