Proteomic Profiling of Sweat Exosome Suggests its Involvement in Skin Immunity  Chang-Xian Wu, Zheng-Fei Liu  Journal of Investigative Dermatology  Volume 138, Issue 1, Pages 89-97 (January 2018) DOI: 10.1016/j.jid.2017.05.040 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Isolation and identification of sweat exosomes. (a) Flow chart showing sweat exosome purification procedure based on filtering and serial ultracentrifugation. (b) Electron micrograph of negatively stained sweat exosomes. Scale bar = 200 nm. (c) Plot of exosome size distribution evaluated by dynamic light scattering. The plot shows a Gaussian distribution profile with a mean peak at 78 ± 8 nm. h, hour; min, minute; PBS, phosphate buffered saline. Journal of Investigative Dermatology 2018 138, 89-97DOI: (10.1016/j.jid.2017.05.040) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Immunoblots of proteins in human sweat exosomes. (a) 12.5% SDS-PAGE of sweat exosome for LC-MS/MS. (b) Western blot of sweat exosome and 293T cell control. Immunoblots were recognized by specific antibodies against lactoferrin, ALIX, HSP70, CD63, and α-tubulin. The results depicted in Figure 2b are representative of three independent experiments (n = 3). LC, liquid chromatography; M, unstained protein molecular weight marker; MS, mass spectrometry; SE, sweat exosome. Journal of Investigative Dermatology 2018 138, 89-97DOI: (10.1016/j.jid.2017.05.040) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Confirmation of removal of potential contamination during sweat exosome purification. (a) Western blot of sweat, sweat exosomes, HaCaT cells, processed HaCaT cells, skin scrape, and processed skin scrape. Immunoblots were recognized by specific antibodies against α-tubulin and CD63. HaCaT cells were lysed at 95 °C for 15 minutes, and the lysate was treated with the protocol for sweat exosome isolation. The resulting product was called processed HaCaT cell. (b) Sweat exosomes, sweat, processed skin scrape, and skin scrape were tested for the presence of bacteria by PCR amplification of 16S rRNA. Sterilized distilled water and E. coli DH5α served as negative control and positive control, respectively. Journal of Investigative Dermatology 2018 138, 89-97DOI: (10.1016/j.jid.2017.05.040) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Functional classification of the sweat exosome proteins from Gene Ontology annotation. Histograms represent the assigned classification of (a) cellular localizations, (b) molecular functions, and (c) biological process. Journal of Investigative Dermatology 2018 138, 89-97DOI: (10.1016/j.jid.2017.05.040) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Venn diagram of sweat exosomes. (a) Venn diagram of total proteins from sweat exosomes and sweat. Numbers represent the distinct proteins in the respective overlapping and non-overlapping areas. (b) Venn diagram of total proteins from sweat, saliva, urine, and plasma exosomes, showing common and unique proteins. Journal of Investigative Dermatology 2018 138, 89-97DOI: (10.1016/j.jid.2017.05.040) Copyright © 2017 The Authors Terms and Conditions