Fig 2: Effect of rh-NTN4 on neurite outgrowth in human iPSC-derived sensory neurons (A–C). Optical microscopy images at day 4 after rh-NTN4 stimulation: (A) vehicle-treated cells, (B) cells treated with 50 ng/mL rh-NTN4, (C) cells treated with 500 ng/mL rh-NTN4. (D) Quantification of neurite length in vehicle-treated, 50 ng/mL rh-NTN4-treated, and 500 ng/mL rh-NTN4-treated cells at day 4. Data are presented as mean ± SE. (E–G) Fluorescence microscopy images at day 14 after rh-NTN4 stimulation: (E) vehicle-treated cells, (F) cells treated with 50 ng/mL rh-NTN4, (G) cells treated with 500 ng/mL rh-NTN4. (H) Quantification of neurite length in vehicle-treated, 50 ng/mL rh-NTN4-treated, and 500 ng/mL rh-NTN4-treated cells at day 14. Data are presented as mean ± SE. * indicates significant differences between groups (p < 0.05). (I–K) Neurite length per neuron in the (E–G) images was measured using NeuronJ, an ImageJ plug-in.

Fig 3: Relationship between NTN4 expression and osteoarthritis pathology.

Fig 4: Expression of NTN4 and its receptors in iPSC-derived sensory neurons (iPSC-SNs) and synovial fibroblasts. Box-and-whisker plots showing the relative expression levels of (A) NTN4, (B) UNC5B, and (C) NEO1 in fibroblasts (Fb), normalized to the expression levels in iPSC-derived sensory neurons (iPSC-SNs), which are set as 1. An asterisk (*) indicates statistical significance with p-values less than 0.05, demonstrating differences between fibroblast and iPSC-SN expression levels.

Fig 5: Flow cytometric analysis of vehicle- and recombinant Netrin-4-treated fibroblastic cells derived from the synovium. (A–C): Dot plot analysis of fibroblastic cells treated with vehicle (A), 50 ng/mL (B), and 500 ng/mL (C) recombinant human Netrin-4 (rh-NTN4). (D) Mean fluorescence intensity of vehicle- and rhNTN4-treated fibroblastic cells displayed as box-and-whisker plots. * indicates significant differences (p < 0.05) vs. vehicle.