Fig 2: CD107a immunohistochemical staining in human fetal skeletal muscle.CD107a positive staining appears red, while CD31 positive staining appears black.

Fig 3: Adipoprogenitor cell identity of CD107ahigh human mesenchymal cells and correlation to exocytosis during early adipogenic differentiation.(A–G) CD107alowCD31-CD45- and CD107ahighCD31-CD45- cells derived from human subcutaneous WAT were exposed to adipogenic differentiation conditions. (A,B) Adipocyte colony formation frequency (CFU-AD) detected in human CD107alow and CD107ahigh cells. Whole well images shown. (C,D) Oil red O (ORO) staining and photometric quantification at d7 of adipogenic differentiation among human CD107alow and CD107ahigh cells. Representative whole well and high magnification images shown. (E–G) Adipogenic gene expression among human CD107alow and CD107ahigh cells by qRT-PCR at d 3, 7, and 10 of differentiation, including (E) Peroxisome proliferator-activated receptor-? (PPARG), (F) Lipoprotein lipase (LPL), and (G) Fatty acid-binding protein 4 (FABP4). (H) Immunocytochemical staining of membranous CD107a in the presence of growth medium (GM) or adipogenic differentiation medium (ADM) after 3 d using human, culture-defined adipose-derived stem cells (ASCs). CD107a immunoreactivity appears red, nuclear counterstain appears blue. (I) Photographic quantification of membranous CD107a immunofluorescence under GM or ADM conditions. (J–L) Induction of membranous CD107a expression after adipogenic differentiation across cell types, including (J) culture-defined human ASCs, (K) FACS-purified human perivascular stem cells (PSC), and (L) culture-defined human BMSCs, assessed by flow cytometry after 3 d under GM or ADM conditions. (M,N) Trafficking of CD107a to the cell surface during adipogenesis was inhibited after treatment with Vacuolin-1 (Vac-1, 1 µM), assessed by CD107a immunostaining (M) and flow cytometry (N) after 3 d under GM and ADM conditions. The cell membrane was labeled using Wheat Germ Agglutinin Conjugates (red), while overlap with CD107a immunostaining appears yellow, and DAPI nuclear counterstain appears blue. (O) Dimensional reduction and unsupervised clustering of human stromal vascular fraction (SVF) adipogenic lineage from subcutaneous WAT revealed three cell groups. (P) Trajectory analyses of human SVF adipogenic lineage, colored based on their unsupervised clustering identity. DPP4 (early) and GGT5 (late) expression were used to identify trajectory origin. (Q) Pseudotemporal cell ordering along differentiation trajectories. Pseudotime is depicted from red to purple. (R) Expression heatmap across pseudotime of genes associated with exocytosis. (S) Combined, normalized expression of exocytosis genes shows enrichment (>1) in early progenitors (green shaded area), while more differentiated cells show reduced average expression. Dots in scatterplots represent values from individual wells, while mean and one SD are indicated by crosshairs and whiskers. In column graphs, mean values and one SD are shown. *p<0.05; **p<0.01; ***p<0.001. Statistical analysis was performed using a two-tailed Student t-test (B,D,I–L) or two-way ANOVA followed by Sidak’s multiple comparisons test (E–G,N). Experiments performed in at least biologic triplicate. Black scale bar: 100 µm; white scale bar: 20 µm.

Fig 4: Perivascular CD107a expression typifies a subset of perivascular cells within human subcutaneous white adipose tissue (WAT).Immunofluorescent staining of CD107a (green) and CD34 (red) in human adipose tissue. (A) Tile scan. (B) Larger artery in cross-section, including (B1) outer tunica adventitia, (B2) inner tunica adventitia, and (B3) tunica media and intima. (C) Larger vein in cross-section, including (C1) high magnification of vessel wall. (D) Smaller caliber artery in cross-section, including (D1) high magnification of vessel wall. (E) Capillary in longitudinal cross-section, and (E1) high magnification. (F) Representative FlowJo plot to demonstrate partitioning CD107alowCD31-CD45- and CD107ahighCD31-CD45- fractions from human stromal vascular fraction (SVF). Frequency of CD107alow/high cells across samples is shown in Supplementary file 1 (N = 8 samples). (G) Confirmatory immunofluorescent staining of FAC-sorted CD107alow and CD107ahigh mesenchymal cells (CD31-CD45-PI- cells). (H,I) Representative flow cytometry analysis of freshly isolated CD107alow and CD107ahigh mesenchymal cells, including CD107a, CD34, and CD146. Frequency of expression is shown in relation to isotype control (blue vs. red lines). Frequency of CD34+ and CD146+ cells across all samples is shown in Supplementary file 2, 3 (N = 4 samples). Scale bars: 500 µm (A), 50 µm (B,C,G) and 10 µm (B1–B3,C1,D,D1,E,E1).

Fig 5: CD107a immunohistochemical staining in human atherosclerotic vessel.CD107a positive staining appears red. Red scale bar: 200 µm; black scale bar: 50 µm.