Fig 1: Bladder VEGFA instillation led to an up-regulation of TRPA1 in lumbosacral DRG.(A). Representative data of immunofluorescence labeling of TRPA1 channel in lumbosacral DRG. Scale bar: 20 µm. (B). Normalized signal intensity of TRPA1 antibody in lumbosacral DRG isolated from VEGFA-instilled animals is significantly higher than in ganglia from saline-instilled animals. (C). Representative data of immunofluorescence labeling of TRPV1 channel in lumbosacral DRG. Scale bar: 20 µm. (D). Normalized signal intensity of anti-TRPV1 is not significantly different in lumbosacral DRG isolated from VEGFA-instilled animals when compared to those from saline-instilled animals. (E). Representative data of immunofluorescence labeling of VEGFR1 in lumbosacral DRG. Scale bar: 20 µm. (F). Normalized signal intensity of anti-VEGFR1 in lumbosacral DRG isolated from female (left) and male (right) mice. No significant difference in anti-VEGFR1 signal was detected between saline- and VEGFA-instilled animals. (G). Representative data of immunofluorescence labeling of VEGFR2 in lumbosacral DRG. Scale bar: 20 µm. (H). No significant difference in anti-VEGFR2 signal was detected between saline- and VEGFA-instilled animals.
Fig 2: Expression of Sox2 and TRPA1 in the corpus callosum shown by immunofluorescence and WB. WB assays indicate the expression of Sox2 (A), TRPA1 (C) and ß-actin (loading control) in the corpus callosum and hippocampus of mice in different groups. Double immunofluorescence staining for PDGF-Ra+ Sox2+ and GFAP+ Sox2 + (B), and oligo2+ TRPA1+ and GFAP+ TRPA1+ (D) in the corpus callosum of mice. Data are presented as mean ± SEM. N = 3 per experimental group; experiment repeated two times. Scale bars = 50 µm. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus the CPZ group.
Fig 3: TRPA1 enhanced the expression of ß-catenin promoting phagocytosis in HaCaT cells. (A) The protein levels of TRPA1 and ß-catenin were measured by Western blot in HaCaT cells, which were treated with UVA or UVB exposure, JT010 (1 µM, TRPA1 agonist), or overexpressed with HA-TRPA1 respectively. (B and C) Quantification of TRPA1 and ß-catenin protein expression levels. Values are mean value of protein expression normalized to ß-actin and relative to control from three independent tests ± SD. (D) The protein levels of ß-catenin were measured by Western blot in HaCaT cells, which were treated with XAV-939 (10 µM, decreasing ß-catenin expression) alone or before exposure to UVA/UVB. (E) Quantification of ß-catenin protein expression levels. Values are mean value of protein expression normalized to ß-actin and relative to control from three independent tests ± SD. (F) Quantification of fluorescence intensity indicating the fluorescent microspheres uptake was detected by Flow cytometry in HaCaT cells. Values are expressed as mean ±SD from three independent tests. (G) Fluorescent microspheres uptake was observed by fluorescence confocal microscopy, with red fluorescence indicating microspheres, and green fluorescence indicating cell membrane stained with DiO. HaCaT cells were treated with XAV-939 (10 µM) alone or before exposure to UVA/UVB respectively. Scale bar: 50 µm. (H) Quantification of fluorescent microspheres per HaCaT cell. Values are expressed as mean ± SD from three independent tests. (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig 4: Bladder VEGFA instillations caused changes in mRNA expression in TRPA1 and nNOS genes in lumbosacral DRG.Significantly higher TRPA1 mRNA expression and lower nNOS mRNA expression were detected in lumbosacral DRG isolated from VEGFA-instilled mice when compared to saline-instilled mice. No differences were detected in the gene expression of either VEGF receptors.
Fig 5: TRPA1 channels regulated UVA/UVB-induced Ca2+ responses in HaCaT cells. (A) Fluo-4 AM fluorescence intensity indicating calcium concentration was detected by flow cytometry in HaCaT cells after exposure to UVA (225 mJ/cm2) or UVB (25 mJ/cm2) with or without retinal preincubation (12 µM). (B and C) Quantification of the fluorescence intensity at different treatments. Values are mean value of relative fluorescence intensity normalized to control from three independent tests ±SD. (D) Calcium imaging was observed by fluorescence confocal microscopy after treatment with UVA or UVB exposure, JT010 (1 µM, TRPA1 agonist), HC-030031 (10 µM, TRPA1 antagonist), or HA-TRPA1. (Fluo-4 AM, green fluorescence). (**p < 0.01; ***p < 0.001).
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