Fig. 7: Tspan-8 d234-237 is arrested in the ER, sequesters Stx2 and inhibits mucin5-AC·GFP secretion.

a Optical plane from a confocal image of live cells expressing Tspan-8·GFP and labelled with CellBrite® to visualize the plasma membrane. Top row are cells expressing WT Tspan-8 (clone 3) and lower row are cells expressing the truncated version of Tspan-8 d234-237 (clone 8). Arrows point to the plasma membrane. Scale bar is 5 µm. b Quantification of the PCC between Cellbrite® and Tspan-8·GFP. Each gray dot represents the PCC of 1 image with at least 3 cells. n = 10. The red dot is the mean value of the gray dots +/− the standard deviation. The p value was calculated with an ANOVA test. c Optical plane from live cells expressing Tspan-8·GFP at endogenous levels and transiently transfected with Stx2·RFP. Top row are cells expressing WT Tspan-8·GFP (clone 3) and lower row are cells expressing the truncated version of Tspan-8 d234-237 (clone 8). Images of clone 3 (n = 8) and clone 8 (n = 11) were taken from two independent cell cultures. All images showed similar results. Arrows point to the plasma membrane. Arrowheads point to Stx2·RFP trapped in the ER. Scale bar is 5 µm. d Lysates of HT29-N2 cells genetically modified by CRISPR/Cas9 to express WT (clone 3) or a truncated version (clone 8) of Tspan-8·GFP were processed for immunoprecipitation of GFP and western blot analysis. WT HT29-N2 cells were used as a control condition. One of two independent experiment is shown. Both experiments showed similar results. e Representative dot blot showing mucin-5AC secreted by cells expressing WT or a truncated version of Tspan-8. f Quantification of the amount of secreted mucin-5AC in cells expressing the WT or the truncated version of Tspan-8. Each dot represents the signal from one secretion assay. Grouped in different colors are secretion assays that were processed in parallel. n = 12. Red dots represent the mean +/− the standard deviation. One-way ANOVA test: p < 0.001. Source data for b–f are provided as a Source Data file.