Fig 1: FM1-43 fluorescent staining was used to detect the vesicle release in SH-SY5Y neurons for Control group, NGF group, Anti-NGF group, NGF + NC group, and NGF + Atp6v0a1 shRNA group. (a,b) FM1-43 fluorescent staining imaging (×200). (c,d) Comparison of FM1-43 fluorescence intensity (Data are presented as mean ± SD. *: p < 0.05).
Fig 2: The expression level of Atp6v0a1 and CGRP in SH-SY5Y neurons for Control group, NGF group, Anti-NGF group, NGF + NC group, and NGF + Atp6v0a1 shRNA group. (a,b) Real-time polymerase chain reaction results for Atp6v0a1 expression level. (c,d) Enzyme-Linked Immunosorbent Assy (ELISA) results for CGRP expression level (Data are presented as mean ± SD. *: p < 0.05).
Fig 3: The Effects of NGF and Atp6v0a1on CGRP Expression in TG. (a) Lentivirus vector was successfully transduced into trigeminal neurons in vivo. (b) DNA sequencing confirmed the successful recombination of Atp6v0a1 short hairpin RNA (shRNA) sequence and virus vector. (c–e) Comparison of average optical density of the expression levels of CGRP and Atp6v0a1 proteins among Saline + NC group, NGF + NC group, Saline + shRNA group, and NGF + shRNA group in vivo at 3d (c) immunofluorescence imaging (×400). (d) Quantification of fluorescence for CGRP protein. (e) quantification of fluorescence for Atp6v0a1 protein. (f) Comparison of orofacial pain level among Saline + NC group, NGF + NC group, Saline + shRNA group, and NGF + shRNA group through rat grimace scale (RGS). (g–i) Comparison of average optical density of the expression levels of CGRP and Atp6v0a1 mRNA among Saline + NC group, NGF + NC group, Saline + shRNA group, and NGF + shRNA group in trigeminal ganglion at 3d. (g) Fluorescence in situ hybridization imaging (×400). (h) Quantification of fluorescence for CGRP mRNA. (i) Quantification of fluorescence for Atp6v0a1 mRNA (Data are presented as mean ± SD, n = 6. *: p < 0.05).
Fig 4: The expression of CGRP in Trigeminal Subnucleus Caudalis (Vc) for Saline + NC group, NGF + NC group, Saline + Atp6v0a1 shRNA group, and NGF + Atp6v0a1 shRNA group. (a) Immunofluorescence detection of CGRP expression in the superficial layer at 3d (×400). (b) The comparison of CGRP expression level among Saline + NC group, NGF + NC group, Saline + Atp6v0a1 shRNA group, and NGF + Atp6v0a1 shRNA group (Data are presented as mean ± SD, n = 6. *: p < 0.05).
Fig 5: A schematic illustration depicting the mechanisms of NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. The intraganglionic administration of NGF stimulates the upregulation of CGRP in TG is independent on Atp6v0a1, and the upregulation of CGRP in Vc is dependent on vesicle transport.
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