Fig. 1: Sortilin preferably interacts with monomeric thyroglobulin.

a Schematic representation of thyroglobulin (TG) and sortilin (sort) forms used in our experiments. b SDS-PAGE showing binding of his-tagged sortilin ectodomain to endogenous human untagged TG (eTG) by pull-down on Ni-NTA beads. c Size exclusion chromatography (SEC) profile and corresponding SDS-PAGE of his-tagged sortilin preincubated with TG. Sortilin-TG coelution as a monomer-monomer complex (SorTG) is highlighted in yellow d Mass photometry (MP) profile of sortilin preincubated with either mTG (orange) or dTG (grey). The mass of SorTG confirms the interaction of monomeric sortilin with mTG. e Representative confocal images of three independent immunofluorescence experiments: intracellular TG was detectable after 120 min in Nthy-ori 3–1 cells overexpressing full-length sortilin-V5 either in the absence or in the presence of 0.1 mg/mL TG in the cellular medium. TG internalisation depends on the presence of sortilin and it is largely increased when TG is enriched in mTG. Scale bar: 20 µm. f Quantification of TG cellular internalisation. P-values are based on the Kruskal–Wallis test (****p < 0.0001). Mean values ± SD (number of cells = 159 at 0 min, number of cells = 76 for eTG at 120 min, number of cells = 87 for dTG at 120 min, number of cells = 113 for mTG at 120 min). Experiments were performed in triplicate n = 3. A.U.: arb units g MP profiles of recombinant TG (rTG, purple) and iodinated rTG (i-rTG, black), showing that iodination does not affect TG oligomerisation state. h Percentage of SorTG complex formed in vitro (circles) and of TG internalised in cells (squares) plotted against the content of TG monomer present in each sample. The plot suggests a correlation between these two parameters. TG internalised by cells was detected by immunofluorescence assays, measuring the average of TG signal mean intensity for each cell, normalised at t = 0 min (background). t = 0 corresponds to the time point in which the cells are not exposed to TG (right before adding it in the medium). The TG signal detected at this time is considered as unspecific background. All the following TG internalisation signal values were normalised with respect to this time point.