Extended Data Fig. 10: SM treatment cannot rescue the formation of SMS2 foci and migrasome formation in cells expressing SMS2-S217A mutant.
a, The SMS2 model predicted by AlphaFold is coloured by the confidence score. b, Western blot analysis to estimate the protein level of GFP-tagged SMS2 in L929 cells and L929 cells expressing WT SMS2–GFP or the mutants SMS2-S217A–GFP and SMS2-H229A–GFP. c, L929 cells expressing WT SMS2–GFP or the mutant SMS2-H229A–GFP were stained with mCherry–NT-Lys and observed by TIRF microscopy. Scale bars, 10 µm (main images) and 2 µm (magnifications). The boxed areas in the ‘Merge’ panels have been enlarged (right). d, L929 cells expressing WT SMS2–GFP or the mutant SMS2-S217A–GFP were supplemented with vehicle or 5 µM SM. After a 12-h incubation, the cells were stained with mCherry–NT-Lys and observed by TIRF microscopy. Scale bar, 10 µm. e, Statistical analysis of SMS2 foci per cell area in cells from Extended Data Fig. 10c. Data are from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. P values were calculated using a two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant. SMS2 + Vehicle (n = 172 cells) versus SMS2 + SM (n = 166 cells), *P = 0.0316; S217 + Vehicle (n = 157 cells) versus S217 + SM (n = 166 cells), ***P = 0.0007. f, L929 cells expressing WT SMS2–GFP or the mutants SMS2-S217A–GFP were supplemented with vehicle or 5 µM SM. After a 12-h incubation, the cells were stained with mCherry–NT-Lys and observed by confocal microscopy. Scale bar, 10 μm. g, Statistical analysis of the migrasome numbers in the cells from Extended Data Fig. 10e; n = 100 cells per group from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. P values were calculated using a two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant. SMS2 + Vehicle versus SMS2 + SM, P = 0.2875; S217A + Vehicle versus S217A + SM, P = 0.2182; ns, non-significant.
