Sigenome pool

Human Caco-2, A431 or HaCAT cells were cultured as previously stated (Fletcher et al., 2015 (link); Elbediwy et al., 2012 (link)). All siRNA transfections were performed using Lipofectamine RNAiMax transfection reagent (Invitrogen). Briefly, cells were seeded in 6-well plates and treated with the siRNA/transfection mix 2 h post seeding. A final concentration of 50-100 nM siRNA was used for transfections. The following day, another transfection was performed before the cells were trypsinised 4 h later and reseeded either for 2D or 3D culture. 2D siRNA treatments were left for a total of 72 h and 3D treatments were left for a total of 120 h. 3D cultures were prepared as previously stated (Elbediwy et al., 2012). siRNAs were used as siGenome pools (Dharmacon).

siGENOME pools, ON-TARGETplus SMARTpools, individual ON-TARGETplus and customer designed (for E6) siRNAs duplexes were purchased from Dharmacon.
For luciferase assay HeLa(GalELK1-A17) cells were grown in phenol red-free DMEM. Cells were lysed by adding home-made 2X Lysis-luciferase substrate buffer 48 hours after transfection. Luminescence was measured using an Orion microplate Luminometer (Berthold) plate reader.

Murine MIN6 β-cells were routinely cultured at 37°C with 5% CO₂ in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS) Gold (PAA Laboratories). For imaging experiments, MIN6 β-cells were plated at a density of 300 000 onto poly-l-lysine-coated coverslips and left overnight to adhere. Cells were then transfected with either mouse PINK1 siRNA or scrambled control siRNA (siGENOME pools, Dharmacon) as per the manufacturer's instructions and incubated for 48 h. One hour prior to imaging, cells were cultured in HEPES-buffered salt solution (HBSS) supplemented with 20 mmol l⁻¹ HEPES and 2 mmol l⁻¹d-glucose.