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a. Schematic of stretched fibroblasts on mast cells through paracrine signaling. Substance P (SP) and 5-hydroxytryptamine (5-HT) released by P815 mast cells were measured after culturing in the supernatant of stretched fibroblasts (5%; 0, 0.5 or 2 Hz; 30 minutes) for 4 hours (n = 6). b. Schematic of mechanical force on P815 cells, co-cultured with fibroblasts. SP and 5-HT levels in the co-culture supernatant after stretching (5%; 0, 0.5 or 2 Hz; 30 minutes) (n = 6). c. Schematic of the Transwell device (left): P815 cells were seeded in the upper chamber, with the lower chamber containing complete medium (Ctrl), conditioned medium from non-stretched fibroblasts (No stretch), or conditioned medium from stretched fibroblasts (Stretch). Images of cell clustering were stained with crystal violet after 8, 12 or 16 hours of incubation (middle). Scale bar: 50 μm. Statistical chart of mast cells under chemotaxis (n = 6) (right). d. Schematic of cytokine levels in the supernatant of fibroblasts after stretching by <t>ELISA</t> <t>(left).</t> <t>NGF/CXCL1/M-CSF/IL-33/SCF</t> levels measured in cell-free supernatants of fibroblasts after stretching (5%; 2 Hz) for 30 minutes (n = 6). e. Immunofluorescence staining of mice skin primary fibroblasts, with and without Y-27632, pre- and post-stretch: phalloidin (red), nucleus (blue), IL-33 or SCF (green). f. Fluorescence intensity statistics of IL-33 (top) and SCF (down) in fibroblasts. For IL-33, Ctrl (n = 507), Stretch (n = 493), Y-27632 with Stretch (n = 452). For SCF, Ctrl (n = 255), Stretch (n = 359), Y-27632 with Stretch (n = 404). Scale bars are 50 μm.
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a. Schematic of stretched fibroblasts on mast cells through paracrine signaling. Substance P (SP) and 5-hydroxytryptamine (5-HT) released by P815 mast cells were measured after culturing in the supernatant of stretched fibroblasts (5%; 0, 0.5 or 2 Hz; 30 minutes) for 4 hours (n = 6). b. Schematic of mechanical force on P815 cells, co-cultured with fibroblasts. SP and 5-HT levels in the co-culture supernatant after stretching (5%; 0, 0.5 or 2 Hz; 30 minutes) (n = 6). c. Schematic of the Transwell device (left): P815 cells were seeded in the upper chamber, with the lower chamber containing complete medium (Ctrl), conditioned medium from non-stretched fibroblasts (No stretch), or conditioned medium from stretched fibroblasts (Stretch). Images of cell clustering were stained with crystal violet after 8, 12 or 16 hours of incubation (middle). Scale bar: 50 μm. Statistical chart of mast cells under chemotaxis (n = 6) (right). d. Schematic of cytokine levels in the supernatant of fibroblasts after stretching by <t>ELISA</t> <t>(left).</t> <t>NGF/CXCL1/M-CSF/IL-33/SCF</t> levels measured in cell-free supernatants of fibroblasts after stretching (5%; 2 Hz) for 30 minutes (n = 6). e. Immunofluorescence staining of mice skin primary fibroblasts, with and without Y-27632, pre- and post-stretch: phalloidin (red), nucleus (blue), IL-33 or SCF (green). f. Fluorescence intensity statistics of IL-33 (top) and SCF (down) in fibroblasts. For IL-33, Ctrl (n = 507), Stretch (n = 493), Y-27632 with Stretch (n = 452). For SCF, Ctrl (n = 255), Stretch (n = 359), Y-27632 with Stretch (n = 404). Scale bars are 50 μm.
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a. Schematic of stretched fibroblasts on mast cells through paracrine signaling. Substance P (SP) and 5-hydroxytryptamine (5-HT) released by P815 mast cells were measured after culturing in the supernatant of stretched fibroblasts (5%; 0, 0.5 or 2 Hz; 30 minutes) for 4 hours (n = 6). b. Schematic of mechanical force on P815 cells, co-cultured with fibroblasts. SP and 5-HT levels in the co-culture supernatant after stretching (5%; 0, 0.5 or 2 Hz; 30 minutes) (n = 6). c. Schematic of the Transwell device (left): P815 cells were seeded in the upper chamber, with the lower chamber containing complete medium (Ctrl), conditioned medium from non-stretched fibroblasts (No stretch), or conditioned medium from stretched fibroblasts (Stretch). Images of cell clustering were stained with crystal violet after 8, 12 or 16 hours of incubation (middle). Scale bar: 50 μm. Statistical chart of mast cells under chemotaxis (n = 6) (right). d. Schematic of cytokine levels in the supernatant of fibroblasts after stretching by ELISA (left). NGF/CXCL1/M-CSF/IL-33/SCF levels measured in cell-free supernatants of fibroblasts after stretching (5%; 2 Hz) for 30 minutes (n = 6). e. Immunofluorescence staining of mice skin primary fibroblasts, with and without Y-27632, pre- and post-stretch: phalloidin (red), nucleus (blue), IL-33 or SCF (green). f. Fluorescence intensity statistics of IL-33 (top) and SCF (down) in fibroblasts. For IL-33, Ctrl (n = 507), Stretch (n = 493), Y-27632 with Stretch (n = 452). For SCF, Ctrl (n = 255), Stretch (n = 359), Y-27632 with Stretch (n = 404). Scale bars are 50 μm.

Journal: bioRxiv

Article Title: Decoding the fibroblast/mast cell signaling pathway of acupuncture

doi: 10.1101/2025.11.20.688774

Figure Lengend Snippet: a. Schematic of stretched fibroblasts on mast cells through paracrine signaling. Substance P (SP) and 5-hydroxytryptamine (5-HT) released by P815 mast cells were measured after culturing in the supernatant of stretched fibroblasts (5%; 0, 0.5 or 2 Hz; 30 minutes) for 4 hours (n = 6). b. Schematic of mechanical force on P815 cells, co-cultured with fibroblasts. SP and 5-HT levels in the co-culture supernatant after stretching (5%; 0, 0.5 or 2 Hz; 30 minutes) (n = 6). c. Schematic of the Transwell device (left): P815 cells were seeded in the upper chamber, with the lower chamber containing complete medium (Ctrl), conditioned medium from non-stretched fibroblasts (No stretch), or conditioned medium from stretched fibroblasts (Stretch). Images of cell clustering were stained with crystal violet after 8, 12 or 16 hours of incubation (middle). Scale bar: 50 μm. Statistical chart of mast cells under chemotaxis (n = 6) (right). d. Schematic of cytokine levels in the supernatant of fibroblasts after stretching by ELISA (left). NGF/CXCL1/M-CSF/IL-33/SCF levels measured in cell-free supernatants of fibroblasts after stretching (5%; 2 Hz) for 30 minutes (n = 6). e. Immunofluorescence staining of mice skin primary fibroblasts, with and without Y-27632, pre- and post-stretch: phalloidin (red), nucleus (blue), IL-33 or SCF (green). f. Fluorescence intensity statistics of IL-33 (top) and SCF (down) in fibroblasts. For IL-33, Ctrl (n = 507), Stretch (n = 493), Y-27632 with Stretch (n = 452). For SCF, Ctrl (n = 255), Stretch (n = 359), Y-27632 with Stretch (n = 404). Scale bars are 50 μm.

Article Snippet: The level of 5-HT, SP, NGF, M-CSF, CXCL1, IL-33, and SCF were analyzed using ELISA kits (E-EL-0033c, E-EL-0067c, E-EL-M0815, E-MSEL-M0105, E-EL-M0018, E-EL-M2642c, and E-EL-M0636c, respectively) from Elabscience, following manufacturer’s instructions.

Techniques: Cell Culture, Co-Culture Assay, Staining, Incubation, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence