Syt1 is necessary for migrasome biogenesis. (A) Confocal images of migrasomes of cells derived from WT and Syt1 knockout L929. Cells are labeled with WGA (cyan). Scale bar, 10 µm. (B) Quantification of the number of migrasomes in WT and Syt1 knockout L929 shown in A. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (C) Western blot analysis of Syt1 and GAPDH in WT and Syt1 knockout L929. (D) Confocal images of migrasomes of cells derived from WT and Syt1 knockdown MiaPaCa2. Cells are labeled with WGA (cyan). Scale bar, 10 µm. (E) Quantification of the number of migrasomes in WT and Syt1 knockdown MiaPaCa2 shown in D. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (F) Western blot analysis of Syt1 and GAPDH in WT and Syt1 knockdown MiaPaCa2. (G) Confocal images of migrasomes of cells from WT and Syt1 knockout L929 with 0.5 μM BAPTA pretreatment and following with/without 1 mM Ca²⁺ rescue. Left panels: Cells cultured overnight without BAPTA pretreatment. Middle panels: Cells underwent BAPTA pretreatment overnight without Ca²⁺ rescue after 6 h. Right panels: Cells experienced BAPTA pretreatment, 6 h after BAPTA pretreatment, Ca²⁺ was added into culture medium to substitute for the medium. Cells were labeled with WGA (green). Scale bar represents 10 µm. (H) Quantification of the number of migrasomes per 100 μm RF length per cell in WT and Syt1 knockout L929 shown in G. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (I) Confocal image of migrasomes of mouse embryonic stem cells treated with N2B27 at D0 and D4. Cells are labeled with WGA (green). Scale bar, 10 µm. (J) The number of migrasomes per cell from images like those shown in I was quantified. Data shown represent the mean ± SEM; P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (K) mRNA levels of Syt1, determined by quantitative PCR analysis, of D0-D5 in mouse embryonic stem cells treated with N2B27. Quantitative PCR data were normalized to Gapdh mRNA level and data are reported as the mean ± SEM. n = 3 independent experiments. (L) Confocal images of migrasomes of cells derived from WT and Syt1 knockout mouse embryonic stem cells treated with N2B27 at D4. Cells are labeled with WGA (cyan). Scale bar, 10 µm. (M) Quantification of the number of migrasomes in WT and Syt1 knockout group shown in L. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (N) Western blot analysis of Syt1 and GAPDH in WT and Syt1 knockout mouse embryonic stem cells treated with N2B27 at D4. (O) Model of the role of Syt1 in migrasome biogenesis. Source data are available for this figure: SourceData F4.