Fig 1: Compressed PdL fibroblasts activate sensory-like SH-SY5Y neurons in a substance P-dependent manner showing increased cFOS expression and calcium influx. (A,B) Immunofluorescent labeling of cFOS [red, upper panel in (A)] and phalloidin-labeling of the actin cytoskeleton [β-ACT, green, upper panel in (A)] in sensory-like SH-SY5Y neurons stimulated by conditioned medium of compressed hPdLFs. Downregulation of TAC1 expression in hPdLFs was achieved by sequence-specific siRNA (TAC1 siR). Blocking of NK1R in SH-SY5Y was performed by adding aprepitant to the conditioned medium. The intensity of cFOS is shown as thermal LUT [lower panel in (A)] and analyzed in relation to the control in (B). (C,D) Fluorescent labeling of the calcium influx [Fluo-4, green, upper panel in (C)] in SH-SY5Y neurons 90 s after start of stimulation with conditioned medium of hPdLFs with the fluorescence intensity shown as thermal LUT [lower panel in (C)]. In (D) time points after stimulation start (0, 30, 60, 90, and 120 s) were analyzed in relation to the respective control condition for each time point. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; */**/*** in relation to control, ##/### in relation to control +CF. §/§§§ in relation to TAC1 siRNA +CF. One-way and two-way ANOVA with post-hoc test (Tukey’s). Scale bar: 25 μm in (A) and 100 μm (C). Results are shown as mean ± SEM with value distribution.
Fig 2: Sensory-like SH-SY5Y neurons are stimulated by compressed PdL fibroblasts in a substance P-dependent manner showing increased neuronal complexity. (A) Validation of the differentiation of SH-SY5Y cells to sensory-like neurons by immunofluorescent stainings of NeuN (green, upper panel) and CGRP (red, lower panel) with NK1R staining (green, lower panel), phalloidin-labeling of the actin cytoskeleton (β-ACT, red, upper panel) and DAPI for staining cell nuclei (blue). (B) Experimental model illustrating the stimulation of sensory-like SH-SY5Y neurons with conditioned medium of compressed hPdLFs. Downregulation of TAC1 expression in hPdLFs was achieved by sequence-specific siRNA (TAC1 siR). Blocking of NK1R in SH-SY5Y was performed by adding aprepitant to the conditioned medium. (C) Quantitative expression analysis of TAC1 level in compressed hPdLFs treated with TAC1 siRNA. (D) Secretion levels of SP by compressed hPdLFs treated with TAC1 siRNA. (E–I) Visualization of the morphology of stimulated SH-SY5Y neurons by phalloidin-labeled (β-ACT, green, lower panel and traced in black, upper panel in E) and DAPI (blue, lower panel) for staining cell nuclei. Analyzed morphological parameters include the number of neurites from cell soma (F), neurite branch points per 100 μm (G) and the length of the longest neurite (H) combined in the cellular complexity, which is shown in relation to the control in (D). */# p < 0.05; **/## p < 0.01, ***/### p < 0.001; */**/*** in relation to control, #/##/### in relation to control +CF. One-way ANOVA with post-hoc test (Tukey’s). Scale bar: 50 μm in (A) and 25 μm (C). Results are shown as mean ± SEM with individual values or distribution.
Fig 3: Mechanically compressed PdL fibroblasts show a duration- and intensity-dependent expression and secretion of TAC1 and substance P. (A) Experimental model illustrating the stimulation of human periodontal ligament fibroblasts (hPdLFs) with a compressive force (CF) of 2 g/cm2 for 6, 24, and 48 h using a sterile glass plate. (B) Quantitative expression analysis of TAC1 in hPdLFs stimulated with increasing durations of CF. The expression levels are displayed in relation to the unstimulated control. (C) Protein expression levels of TAC1 in hPdLFs stimulated with increasing durations of CF. (D) Secretion levels of TAC1-derived neuropeptides, specifically substance P and neurokinin A, detected in the medium of hPdLFs stimulated with increasing durations of CF. (E) Experimental model illustrating the stimulation of hPdLFs with a compressive force of 2 g/cm2, 4 g/cm2 and 6 g/cm2 for 24 h using sterile glass plates. (F) Quantitative expression analysis of TAC1 in hPdLFs stimulated with increasing CF. The expression levels are displayed in relation to the unstimulated control. (G) Secretion levels of TAC1-derived neuropeptides detected in the medium of hPdLFs stimulated with increasing CF. (H) Secretion levels of SP in the medium of hPdLFs stimulated with a CF of 2 g/cm2 for 24 h. */#/§ p < 0.05; **/## p < 0.01; *** p < 0.001; */**/*** in relation to control, #/## in relation to 6 h of 2 g/cm2 CF in (B,D) and 24 h of 2 g/cm2 CF (F,G), §§ in relation to 24 h of 2 g/cm2 CF in (B,D) and 24 h of 4 g/cm2 CF (F,G). One-way ANOVA with post-hoc test (Tukey’s). Results are shown as mean ± SEM with individual values.
Supplier Page from Abcam for Substance P ELISA Kit