Fig. 6.

Retromer-dependent cargo recycling is necessary for copper homeostasis. (A,B) Control and VPS35 KO HeLa cells were incubated with nine concentrations of CuCl₂ ranging from 3.12 to 800 µM for (A) 48 or (B) 72 h. Each concentration was performed in quadruplicate. MTT reduction was measured by absorbance at 595 nm. Each data point represents the mean± s.e.m. absorbance at 595 nm normalised to the baseline reading without addition of CuCl₂; n=4 independent experiments. (C) MTT assays were performed as described above subjecting VPS35 KO HeLa cells expressing GFP or GFP–VPS35 to increasing concentrations of CuCl₂ for 48 h. Each data point represents the mean±s.e.m. absorbance at 595 nm normalised to the baseline reading without addition of CuCl₂; n=3 independent experiments. *P<0.05; **P<0.01; ***P<0.001 (unpaired Student's t-test).