Plxin retroviral system

HeLaM cell populations stably expressing VARP-GFP constructs were generated using the pLXIN retroviral system (Clontech). When indicated, HeLaM cytosol was extracted prior to fixation by rinsing cells briefly with PBS, followed by incubation for 1 min in PBS (with Ca²⁺ and Mg²⁺) containing 0.05% saponin, and then immediately formaldehyde fixed. IF confocal microscopy and western blotting were carried out essentially as described elsewhere (Schäfer et al., 2012 (link)). For quantitative IF confocal microscopy of GLUT1 and LAMP1 colocalization, cell fields (three independent experiments with five fields each per condition, average of ≥8 cells per field) were randomly selected based on nuclear stain and focused using the LAMP1 signal. Single confocal images were acquired corresponding to 1 Airy unit, and the degree of colocalization of two channels was measured by Manders M₁/M₂ colocalization coefficients (Manders et al., 1993 ) using Zen software (Carl Zeiss). To negate the effects of inherent cell-to-cell variability of GLUT1 surface staining, the fraction of LAMP1 signal colocalizing with GLUT1 was measured for each field.

Patients’ dermal fibroblasts were cultured in Minimum Essential Medium Eagle (MEM) (Sigma) supplemented with 10% foetal bovine serum (FBS) (Sigma), 1-mM sodium pyruvate (Sigma), 2 mM Glutamax-1 (Gibco), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified atmosphere of 5% CO₂. Healthy adult and neonatal fibroblasts were obtained from Sigma (catalogue numbers 106-05A; 106-05N). Fibroblasts from a 1-year old patient with Niemann–Pick type C (GM23162) were from Coriell Institute For Medical Research (New Jersey, USA).
HeLaM cells stably expressing HA-tagged VPS33A^WT were generated and cultured as previously described (10 ) and cells stably expressing HA-tagged VPS33A^R498W were generated in the same way, using the pLXIN retroviral system (Clontech).
Cells were incubated with proteasome inhibitors (MG-132 from Sigma or bortezomib from Stratech), or the substrate reduction agents eliglustat tartrate (kindly donated by Professor James Shayman, University of Michigan) or miglustat (Zavesca) donated by patients (incubation times and concentrations as indicated in figure legends).