Fig. 3: Monocytes produce fewer migrasomes in Tspan9^–/– mice, and T9 KO results in a marked reduction of total cytokine levels in the blood.

a Total secretion (TS) analysis of TNF-α and IL-6 in the indicated monocytes as shown in Fig. 2d and e. Representative densitometry analysis of western blot gray values was shown. Three independent experiments were conducted. The ratio of TNF-α or IL-6 in total secretion vs cell body was quantified. Quantification is shown as the means ± SEM from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses (right panel). b Equal numbers of WT and T9 KO monocytes were labeled with anti-CCR2 antibodies conjugated to different colored tags. The color-coded cells were mixed for injection into WT mice, and intravital imaging of the mouse liver was performed. Blood vessels are labeled with WGA-AF488. Time interval, 18 s. Scale bar, 20 μm. The right panel shows statistical analysis of the number of migrasomes per cell. Data are means ± SEM of more than 100 cells from three independent experiments. Two-tailed unpaired t-test was used to compare the datasets. c Diluted whole blood collected from mice with LPS stimulation was stained with CCR2-PE antibody and CD9-APC antibody for 30 min. Imaging flow cytometry analysis was performed to measure the number of monocyte migrasomes (CCR2-positive) and platelets (CD9-positive) in the blood from WT and T9 KO mice. Scale bars, 5 μm. Quantification of monocyte-derived migrasomes and platelets is shown as the means ± SEM. n = 20 mice from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses (right panels). d Imaging analysis of CCR2-positive vesicles isolated from WT and T9 KO mouse blood samples. Scale bar, 10 μm. Quantification of monocyte-derived migrasomes is shown as the means ± SEM. n > 50 fields of view from 12 mice. Two-tailed unpaired t-test was used for statistical analyses (right panel). e After LPS stimulation, monocytes, monocyte-derived migrasomes, and small EVs were purified from mouse blood samples and then subjected to western blot analysis using the indicated antibodies. Lysates of monocyte cell bodies (C), monocyte-derived migrasomes (M), and small EVs (1#, 120,000× g and 2#, 160,000× g) were normalized to equal total protein loading for western blot analysis using the indicated antibodies. CD63, CD81, and Alix are used as exosome markers; Arf6 and Kif23 are used as microvesicle markers; CD9 is used as a platelet or platelet-derived EV marker. Representative densitometry analysis of western blot gray values was shown. Three independent experiments were conducted. f Monocyte-derived migrasomes from the indicated mice with LPS treatment were isolated from equal volumes of blood, respectively, and then analyzed by western blot analysis using the indicated antibodies. CPQ and Itg α5 are used as migrasome markers in circulating monocytes. Representative densitometry analysis of western blot gray values was shown. Three independent experiments were conducted. g Schematic illustration of the procedure for acquiring circulating monocyte-derived migrasomes (M), soluble proteins (S), and total secretion mixtures (TS) from mouse blood samples. h LPS (12 mg/kg) was injected into mice by i.p. injection. After 2 h, monocyte-derived migrasomes, and soluble proteins were isolated from equal volumes of the indicated mouse blood as shown in g. Migrasomes (M), soluble proteins (S), and total secretion mixtures (TS) were normalized with the volumes of mouse blood and subjected to western blot analysis using the indicated antibodies. Itg α5 and CPQ are used as migrasome markers in circulating monocytes. Both membrane-bound (M) and soluble (S) forms of TNF-α were detected by western blot analysis. Representative densitometry analysis of western blot gray values was shown. Three independent experiments were conducted.