Cell culture, transfection, and siRNA treatment

The mouse catecholaminergic neuronal cells (CAD cells), generated from a brain tumor of transgenic mice by targeted oncogenesis (kind gift of Hubert Laude; Institut National de la Recherche Agronomique, Jouy-en-Josas, France), were cultured in Opti-MEM GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) including 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep). CAD cells express neuronal properties, but they lack neuronal morphology, thus stay undifferentiated in the presence of serum in their medium. This situation can be reversed by serum starvation, and differentiation can be induced [128]. In this study, we used CAD cells in their undifferentiated state as this condition is optimal to study TNTs [19,125,129–131]. HeLa cells WT, HeLa cells myrlysin-KO and KIF1B-KIF5B double KO [100,132], and HeLa cells stably expressing Gal3-Turquoise were cultured in DMEM-GlutaMAX including 4.5 g/L D glutamine and pyruvate (Gibco) supplemented with 10% of FBS. CAD and HeLa cells were transiently transfected with Gal3-GFP, Arl8b-GFP, fluorescently tagged human histone H2B (H2B-GFP or H2B-mCherry) plasmids (final concentration: 1 μg for 150,000 cells/35mm Ibidi μ-dishes, 3 μg for 800,000 cells/T25 flasks) by Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

To down-regulate Arl8b and TFEB, 30 pmol of unique 27mer siRNA duplex against mouse Arl8b and TFEB (CliniSciences, Nanterre, France and OriGene, Rockville, Maryland, USA, respectively) was introduced to CAD cells in parallel to siRNA scramble (sicontrol) by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions and incubated 60 hours prior to be used in coculture experiments and in western blot analysis.

To induce lysophagy, CAD cells were treated with 1 mM LLOMe for 3 hours.