Journal: Biomolecules

Article Title: Intermethod Characterization of Commercially Available Extracellular Vesicles as Reference Materials

doi: 10.3390/biom16010066

Figure Lengend Snippet: Mean PSD and their uncertainties by cell line. EV PSD was measured in triplicate for each method (AF 4 , Cryo-EM, MRPS, PTA) and then combined into a mean distribution by averaging the three probabilities of each replicate for three cell lines: (( a ), left ) GFP, (( b ), left ) LNCaP, and (( c ), left ) MSC. Mean PSD for AF 4 (black), Cryo-EM (red), MRPS (green), and PTA (blue). Single measurement relative uncertainties for each independent replicate in % at each diameter (nm) for three cell lines: (( a ), center ) GFP, (( b ), center ) LNCaP, and (( c ), center ) MSC for AF 4 (black), Cryo-EM (red), MRPS (green), and PTA (blue). AF 4 single measurement relative uncertainty values are near zero. Replicate-to-replicate relative uncertainties in % at each diameter (nm) for the mean distributions of three cell lines: (( a ), right ) GFP, (( b ), right ) LNCaP, and (( c ), right ) MSC for AF 4 (black), Cryo-EM (red), MRPS (green), and PTA (blue). Cryo-EM was not performed on GFP VLPs. Cryo-EM was not performed in triplicate; a single analysis of ≈500 particles was evaluated.

Article Snippet: An Eclipse DualTec AF 4 separation system (Wyatt Technology, Santa Barbara, CA, USA) was interfaced with an Agilent HPLC system (Model 1260, Agilent Technologies, Santa Clara, CA, USA) including a UV/Vis diode array detector (Model 1260, Agilent Technologies, Santa Clara, CA, USA), a HELEOS-II multiangle light scattering instrument (HELEOS-II, Wyatt Technology, Santa Barbara, CA, USA), and a differential refractive index detector (Optilab T-Rex, Wyatt Technology, Santa Barbara, CA, USA).

Techniques: Cryo-EM Sample Prep

Journal: Biomolecules

Article Title: Intermethod Characterization of Commercially Available Extracellular Vesicles as Reference Materials

doi: 10.3390/biom16010066

Figure Lengend Snippet: Comparison of mean PSD by method. Particle PSD was measured in triplicate for each cell line (LNCaP EVs, MSC EVs, and GFP VLPs) and then combined into a mean distribution by averaging the three probabilities of each replicate using four methods: ( a ) Cryo-EM, ( b ) MRPS, ( c ) PTA, and ( d ) AF 4 . Cryo-Em was not performed on GFP VLP. Cryo-EM was not performed in triplicate; a single analysis of ≈500 particles was evaluated.

Article Snippet: An Eclipse DualTec AF 4 separation system (Wyatt Technology, Santa Barbara, CA, USA) was interfaced with an Agilent HPLC system (Model 1260, Agilent Technologies, Santa Clara, CA, USA) including a UV/Vis diode array detector (Model 1260, Agilent Technologies, Santa Clara, CA, USA), a HELEOS-II multiangle light scattering instrument (HELEOS-II, Wyatt Technology, Santa Barbara, CA, USA), and a differential refractive index detector (Optilab T-Rex, Wyatt Technology, Santa Barbara, CA, USA).

Techniques: Comparison, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

doi: 10.1038/s41467-025-67456-3

Figure Lengend Snippet: a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1 : n = 3 technical replicates, p = 0.0046, Ccl20 : n = 3 technical replicates, p = 0.0059, Cxcl1 : n = 3 technical replicates, p = 0.0020, Cxcl3 : n = 3 technical replicates, p = 0.0258, Cxcl5 : n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3 : n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1 : n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or si Jmjd3 (gray). Jmjd3 : n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3 : p = 0.0184, Timp1 : p = 0.0044, Ccl20 : p = 0.0160, Cxcl1 : p = 0.0371, Cxcl3 : p = 0.0042. n = 3 independent experiments. g , h Scratch assays of primary murine ( n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT ( n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i , j Scratch assays of primary murine ( n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT ( n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for ( a ), ( e ), ( f ) and 1-way ANOVA tests for ( b ), ( d ), ( g – k ) were performed. Data are presented as the mean ± SEM.

Article Snippet: After stimulation, cell free supernatant was collected and analyzed by the University of Michigan Immune Monitoring Shared Resource Core for CCL-20, CXCL-1, CXCL-5 or specific enzyme immunoassay kits for CXCL-3 (Boster Bio) and TIMP-1 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Wound Healing Assay