Figure 2. Depletion of FAM21 causes mis-sorting of SNX27–retromer cargoes to the Golgi apparatus.

(a–d) hTERT-RPE1 cells transfected with the indicated specific or control (siCTL) siRNAs were immunostained for GLUT1 (green) along with LAMP1 (a, red) or GM130 (c, red). Co-localization of GLUT1 with LAMP1 (b) or GM130 (d) was analysed by calculation of Pearson's coefficient. Graphs express means±s.d. (n=3; >30 cells per group). (e) hTERT-RPE1 cells transfected with an siRNA targeting FAM21 were incubated with 10 μg ml⁻¹ of Alexa 568-conjugated Tf for 20 min at 37 °C before fixation. Cells were then immunostained for GM130 and GLUT1. For comparison, representative magnified views of the Golgi were chosen and displayed. (f) Co-localization at the Golgi area among GLUT1, GM130 and Tf were analysed by calculation of Pearson's coefficient. Graphs express means±s.d. (30 cells per group). (g) hTERT-RPE1 cells transfected with the indicated siRNAs were incubated with 0.05% of dimethylsulphoxide or 5 μg ml⁻¹ of Brefeldin A for 5 min at 37 °C before fixation and immunostained for GLUT1 (green), GM130 (red) and Rab11 (grey). Insets are magnified views of the Golgi. (h) hTERT-RPE1 cells stably expressing Myc-β2AR were transfected with the indicated siRNAs and immunostained for Myc epitope (green) and GM130 (red). (i) Cells with Myc signals accumulating at the Golgi from h were counted (n=2; 200 cells per group). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bars, 10 μm. *P<0.05, **P<0.01, ***P<0.001; NS, not significant.