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a pH-responsive <t>DIPMA-MK-3207.</t> b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
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a pH-responsive <t>DIPMA-MK-3207.</t> b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
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a pH-responsive <t>DIPMA-MK-3207.</t> b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
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a pH-responsive <t>DIPMA-MK-3207.</t> b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
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a pH-responsive <t>DIPMA-MK-3207.</t> b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
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a pH-responsive DIPMA-MK-3207. b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice

doi: 10.1038/s41467-022-28204-z

Figure Lengend Snippet: a pH-responsive DIPMA-MK-3207. b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.

Article Snippet: Assemblies of DIPMA-MK-3207 and DIPMA-Ø were dialyzed against PBS for 24 h (MWCO 3500, Membrane Filtration Products).

Techniques: Transmission Assay, Expressing, Incubation, Concentration Assay, Inhibition, Injection