Fig 2: Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).

Fig 3: Localization of transfected EGFP-SDCCAG3 to cilia in IMCD3 cells.IMCD3 cells were transfected with the indicated EGFP-SDCCAG3 expression constructs (green) and stained for the cilia axoneme marker acetylated tubulin (blue) and the basal body marker gamma-tubulin (red). (a) Full-length EGFP-SDCCAG3 co-localized with gamma-tubulin at the basal body. (b) A deletion construct of SDCCAG3 lacking amino acids 1-100 (?1-100) did not co-localize with gamma-tubulin at the basal body. (c) A truncated version of SDCCAG3 comprising only amino acids 1-100 was sufficient for basal body localization of SDCCAG3. Images are representative of three different experiments. Scale bar represents 5µm. (d) A schematic diagram of the transfected EGFP-SDCCAG3 expression constructs showing the full length protein (I), the construct comprising only amino acids 1-100 (II) and SDCCAG3 lacking amino acids 1-100 (III).

Fig 4: Depletion of ENTR1 delays sorting of Fas into intraluminal vesicles. a Immunofluorescence of Fas in cells transfected with constitutively active Rab5 Q79L. Control or ENTR1 no.3 siRNA treated HeLa cells were transfected with Rab5 Q79L-GFP and stimulated with anti-Fas (CH-11) antibody (1 µg/ml) for 1 h in the presence of leupeptin (100 nM). Cells were fixed and observed by a confocal microscope. Insets show representative expanded endosomes. b Example of line scale analysis used for quantifying Fas localised to intraluminal vesicles. The normalised diameter represents the diameter of the endosome, where 0 and 100 correspond to the pixel distances with the highest and second highest pixel intensities, representing the limiting membranes of the endosomes. Blue and red traces represent the normalised fluorescence pixel intensity measured across the endosomes in control and ENTR1 depleted cells, respectively, with the maximum pixel intensity across the line normalised to 100. Region covered by dotted line shows the normalised fluorescence values of pixels from 40–60% of the normalised diameter that were used to determine the mean intraluminal fluorescence for each endosome. c Graphical representation of the compiled results of the line scale analysis for Fas. Middle (40–60%) fluorescence value expressed as a percentage of the limiting membrane (normalised diameter). Unpaired t-test, n > 50 endosomes from three independent experiments, **p < 0.01, p = 0.0075, error bars represents ± s.e.m. d Immunofluorescence analysis of intraluminal sorting of Fas in control or ENTR1 no.3 siRNA treated HeLa cells transfected with constitutively active Rab5 Q79L using 3D-Structured illumination microscopy. e Three-dimensional modelling of endosomes highlighted by the insets in d. Scale bars represent 5 µm (a, d, e)

Fig 5: Co-localization of SDCCAG3 and IFT88 in cells.(a) Co-localization of SDCCAG3 and EGFP-IFT88 at centrosome. HeLa cells were grown on coverslip and transiently transfected with an expression construct for EGFP-IFT88. After 20 hours, cells were fixed and immunostained for SDCCAG3 and pericentrin (PCNT). Arrows indicate the co-localization at centrosomes. Scale bar represents 5 µm. (b) Localization of SDCCAG3 to the basal body is independent of IFT88. IMCD3 cells treated with scrambled or mouse IFT88 siRNA and transfected with EGFP-SDCCAG3 were stained with gamma-tubulin antibody. Scale bar represents 5 µm. (c) Quantification of co-localization of EGFP-SDCCAG3 with gamma-tubulin in cells treated with the indicated siRNA; n = 3, unpaired t-test, error bars ± s.e.m, ns = non-significant. (d) Immunoblot analysis validating knockdown of IFT88 in mIFT88 siRNA treated IMCD3 cells. Cropped blots are shown, full-length blots are presented in supplementary figure S6.