Supplementary Figure 1: Tspan4 is necessary for migrasome formation.

a, mRNA levels of Tspan4 in Tspan4-GFP-expressing NRK cell lines and their parent cells were measured by real-time PCR. Data shown represent mean±SEM (n=3 independent experiments). b, Cells with stable Tspan4-GFP expression were treated with or without deglycosylation mix, then lysed and analyzed by western blot. c, mRNA levels of 9 TSPAN genes in the MGC-803 cell line were measured by real-time PCR and the values were normalized by the TSPAN4 mRNA level. Data shown represent mean±SEM (n=3 independent experiments). d, Verification of the NRK Tspan4 knockout cell line by PCR. e, The average number of migrasomes per 100 µm RF in wild type and Tspan4-KO NRK cells. Data shown represent mean±SEM, n=100 cells for each group. ***P<0.001. f, Statistical analysis of migrasome numbers in wild type L929 and Tspan4-KO L929 cell lines. Data shown represent mean±SEM; n=173 cells for WT L929; n=168 cells for Tspan4-KO L929. NS= No significance. Unprocessed blots related to b were presented in Supplementary Fig. 6. Experiments related to a-f were independently repeated 3 times. The quantifications in e and f are pooled from these independent experiments. Two-tailed unpaired t-test method was used for statistical analysis. The statistical details for a, c, e and f were available in Supplementary Table 4.