bSMase-induced changes in backbone fluorophores are absent in expanded samples. Samples were incubated with TFSM 1 for 24 h. Then, cells were washed, treated with bSMase, and either embedded in Mowiol or in the hydrogel, which was left unexpanded or 4x expanded. Scale bars: 40 m. The mean fluorescence was measured within cell-containing areas ( g ), and fold changes were calculated (green bars, right y -axis). n = 5 for gelated + 100 ng/ml bSMase and gelated + expanded + 100 ng/ml bSMase, otherwise n = 6. Statistics: Two-sided unpaired Student’s t test ( b , c ). Two-way ANOVA and Šídák’s multiple comparisons test ( d ), two-sided one-sample t test (e), mixed effects analysis, and Šídák’s multiple comparisons test ( f , g ). Bars represent means ± SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.
