Journal: Advanced Science

Article Title: Osteoclast‐Derived SLIT3 Mediates Osteoarthritis Pain and Degenerative Changes

doi: 10.1002/advs.202517545

Figure Lengend Snippet: Pain in TRAP‐Cre; Slit3 −/− mice is reduced. a) Experimental design for TRAP‐Cre transgenic mice injected with AAV9‐Slit3‐RNAi. b) Immunofluorescence of SLIT3 (red) in condyles. The white dashed line represents the boundary between subchondral bone and cartilage. Scale bars:10 µm. c) Quantitative analysis in panel (b). n = 6. d) Representative results of OFT and EPM. e,f) Quantitative analysis of Von Frey experiment (e), OFT and EPM (f). n = 6. Statistical analyses were performed using Student's t ‐test in (c), and one‐way ANOVA with Holm–Šidák multiple comparison tests in (e,f). ** p < 0.01. *** p < 0.001. **** p < 0.0001. ns : no significance.

Article Snippet: To evaluate the axonal morphology of the TGs in the different groups (SLIT3 protein, RPD353Mu01, Cloud‐clone, USA; Neutralizing antibodies against SLIT3, AF3629, R&D Systems, USA), immunofluorescence labeling (β3‐tubulin, 1:300, 2144, Cell Signaling Technology) and crystal violet staining ( n = 6) were conducted.

Techniques: Transgenic Assay, Injection, Immunofluorescence, Comparison

Journal: Advanced Science

Article Title: Osteoclast‐Derived SLIT3 Mediates Osteoarthritis Pain and Degenerative Changes

doi: 10.1002/advs.202517545

Figure Lengend Snippet: Immunofluorescence imaging and analysis of sensory nerve distribution in the condyle of Slit3 ‐knockdown mice. a) Representative images of immunofluorescence staining for PGP9.5 (red), CGRP (green) and DAPI (blue) along the TMJ osteochondral junction. Scale bars: 10 µm. b) Quantitative analysis of the density of PGP9.5 + nerve fibers and CGRP + nerve fibers in the TMJ osteochondral junction. n = 6. c) Schematic diagram of anterograde tracing. d) Representative images of the condylar sensory nerve. EGFP (rAAV‐CAG‐EGFP‐WPRE‐hGH pA, green), DAPI (blue). Scale bars: 10 µm. e) Quantitative analysis of the density of sensory nerve fibers. n = 6. The white dashed line represents the boundary between bone and cartilage. Statistical analyses were performed using one‐way ANOVA with Holm–Šidák multiple comparison tests. **** p < 0.0001. ns : no significance.

Article Snippet: To evaluate the axonal morphology of the TGs in the different groups (SLIT3 protein, RPD353Mu01, Cloud‐clone, USA; Neutralizing antibodies against SLIT3, AF3629, R&D Systems, USA), immunofluorescence labeling (β3‐tubulin, 1:300, 2144, Cell Signaling Technology) and crystal violet staining ( n = 6) were conducted.

Techniques: Immunofluorescence, Imaging, Knockdown, Staining, Anterograde Tracing, Comparison

Journal: Advanced Science

Article Title: Osteoclast‐Derived SLIT3 Mediates Osteoarthritis Pain and Degenerative Changes

doi: 10.1002/advs.202517545

Figure Lengend Snippet: Slit3 is specifically knocked out in osteoclasts of Slit3 ‐CKO mice. a) Schematic diagram of Slit3 ‐CKO mice. b) Immunofluorescence of SLIT3 (green) and CD51/61 (red) in subchondral bone. The white dashed line represents the boundary between subchondral bone and cartilage. Scale bars:10 µm. c) Quantitative analysis in panel (b). n = 6. d) qRT‐PCR analysis of the gene expression of Slit3 in subchondral bone in the two groups. n = 3. e,f) Western blot and its quantitative analysis, g) ELISA, and h,i) immunohistochemical staining and quantitative analysis of SLIT3 expression in subchondral bone in the WT and Slit3 ‐CKO groups. n = 3. Scale bars: 70 µm. Statistical analyses were performed using Student's t ‐test. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p <0.0001. ns : no significance.

Article Snippet: To evaluate the axonal morphology of the TGs in the different groups (SLIT3 protein, RPD353Mu01, Cloud‐clone, USA; Neutralizing antibodies against SLIT3, AF3629, R&D Systems, USA), immunofluorescence labeling (β3‐tubulin, 1:300, 2144, Cell Signaling Technology) and crystal violet staining ( n = 6) were conducted.

Techniques: Immunofluorescence, Quantitative RT-PCR, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Expressing

Journal: Journal of translational medicine

Article Title: SLIT3-mediated intratumoral crosstalk induces neuroblastoma differentiation via a spontaneous regression-like program.

doi: 10.1186/s12967-025-06621-0

Figure Lengend Snippet: Fig. 6 PLCβ/PKC signaling mediates SLIT3-induced neuroblastoma cell differentiation. A Volcano plot displaying DEGs comparing cells treated with or without 200 ng/mL SLIT3 recombinant protein for 72 h. Upregulated genes are colored in yellow and downregulated genes are colored in green. B GSEA on the MSigDB database revealed significant enrichment of SLIT-ROBO signaling gene sets and mature neuronal synaptic function gene sets upon SLIT3 treatment C GSEA analysis shows top 10 enriched pathways from Reactome database in SLIT3-treated group compared with Ctrl group. Upregulated and downregulated pathways are colored in yellow and green, respectively. D PLCβ/DAG/IP3 signaling-related gene sets enriched in Stage 4S sample cells compared with Stage 4 sample cells within cluster cSHR (upper panel), and consistently enriched in cluster cIVS compared with cluster cIV (lower panel). E Chord diagram showing gene composition of PLCβ/DAG/IP3 signaling-related gene sets. F Heatmap showing expression of common genes from PLCβ/DAG/IP3 signaling-related gene sets in SLIT3-treated versus Ctrl groups. G A brief overview of PLCβ/DAG/IP3/PKC signaling cascade. Upon activation, membrane-bound PLCβ catalyzes PIP2 hydrolysis into DAG and IP3. DAG directly activates PKC, while free IP3 binds to IP3R receptors on endoplasmic reticulum membrane, triggering calcium release and further activating PKC. H Western blot analysis of neuronal differentiation markers pTrkA, MAP2, SNAP25 in SK-N-BE(2) cells treated with 1 μM U-73122, or 2 μM Ro 31–8220 for 72 h in the presence of Ctrl or SLIT3 conditioned medium. I RT-qPCR analysis of neuronal differentiation markers GAP43, TH, and ENO2 mRNA expression in SK-N-BE(2) cells treated with 1 μM U-73122, or 2 μM Ro 31–8220 for 72 h in the presence of Ctrl or SLIT3 conditioned medium. Data were presented as mean ± SD (n = 3, one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: The following primary antibodies were used: rabbit anti-pTrkA (PTMBio), rabbit anti-MAP2 (Proteintech), mouse anti-SNAP25 (PTMBio), rabbit anti-SLIT3 (Affinity), mouse antiFLAG (Sigma-Aldrich) and mouse anti-GAPDH (Proteintech).

Techniques: Cell Differentiation, Recombinant, Expressing, Activation Assay, Membrane, Western Blot, Quantitative RT-PCR