S0615

HeLa p62 KO cells were cultured in Eagle’s minimum essential medium with 10% fetal bovine serum (Biochrom AG, S0615), nonessential amino acids, 2 mM L-glutamine, and 1% streptomycin–penicillin (Sigma, P4333). For transfection the same media was used but without 1% streptomycin–penicillin. Cells were fixed in 4% PFA for 20 min at room temperature. For immunostaining, cells were permeabilized with cold methanol for 5 min at room temperature, blocked in 3% goat serum/PBS, and incubated at room temperature with antibodies. For DNA staining 1:4000 dilution was used in PBS of DAPI (Thermo Fisher Scientific; pr.66248). Samples were mounted using Mowiol 4-88 (Calbiochem 475904). Cells were examined using a Zeiss LSM780 or LSM800 microscope with a 63 × 1.4 oil objective or a Leica TCS SP8 confocal microscope, 40 × 1.3 oil objective.

The human epithelial colorectal adenocarcinoma cell line purchased from ATCC was used in this study (Caco-2, ATCC^® HTB-37TM, Manassas, VA, USA). The colon cancer cell line was maintained in complete growth media of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Biochrom, S0615), 2 mM L-glutamine, 0.5% penicillin/streptomycin (Sigma, P4333) and 0.5% amphotericin B (Sigma, A2942). Cultured cells were incubated at 37 °C in 5% CO₂ and 85% humidity.

In-cell NMR samples were prepared following a recently developed electroporation protocol37 . Briefly, A2780 and RCSN-3 cells were grown at 37 °C, 5% CO₂, in T175 culture flasks in RPMI 1640 (Millipore, FG1215) and DMEM-Ham's F-12 (Biochrom, FG4815) growth media, respectively, supplemented with 10% fetal bovine serum (Biochrom, S0615) until 80% confluence. In all, 50–100 × 10⁶ cells were collected for each in-cell NMR sample. Cells were detached with mild trypsin treatment (Biochrom, L2153) and resuspended in 2 ml of electroporation buffer (100 mM sodium phosphate, 15 mM HEPES, 5 mM KCl, 15 mM MgCl₂, 2 mM ATP and 2 mM reduced glutathione, pH 7.0). Fully oxidized, uniformly ¹⁵N isotope- or ¹⁵N methionine-enriched α-Syn (500 μM) was added to electroporation mixtures and aliquots (100 μl) were used for individual electroporations with an Amaxa nucleofector (Lonza) according to the manufacturer's instructions. Cells were re-seeded in 15-cm culture dishes and allowed to recover for 4 h at 37 °C (CO₂ incubator). Cells were washed 3 × with pre-warmed PBS and collected with mild trypsin treatment, washed 2 × with pre-warmed growth media to remove excess trypsin and resuspended in pH-stable Leibovitz's L-15 medium (Gibco, 31415-029) supplemented with 10% D₂O. Finally, cells were transferred to NMR tubes for in-cell NMR experiments.

Single-cell suspensions of iPSCs or iPSC-derived cardiomyocytes were used for flow cytometry. iPSCs were blocked with 5% FBS (Biochrom, S0615) in PBS and stained. Cardiomyocytes were fixed in Roti®-Histofix 4% (Roth, P087) for 20 min at 4 °C and transferred to FACS buffer, containing 5% FBS, 0.5% saponin (Sigma-Aldrich, 47036), and 0.05% sodium azide (Sigma-Aldrich, 71290) in PBS. Samples were analyzed with a BD FACSCanto™ II Flow Cytometer and the BD FACSDiva™ Software 6.0 or BD FlowJo™ V10. In brief, cells were selected by gating SSC-A vs. FSC-A. Then, single cells were gated with FSC-H vs. FCS-A, before gating for fluorescence intensity using isotype-stained cells as control.

The epithelial-like human breast adenocarcinoma cell line Michigan Cancer Foundation-7 (MCF- 7, DSMZ no. ACC 115), known to express claudin-3, -4, and -7 (Kominsky et al., 2003 (link); Todd et al., 2015 (link)) was used as claudin positive cells. For controls, we used a breast cancer cell line, MDA-MB-231 with minimal expression of claudin -3, -4, and -7 (Becker et al., 2018 (link)). Between experiments, the cells were kept in an incubator at 37°C with 5% CO₂. Cells were cultured in DMEM/F12 (F4815, Biochrom) supplemented with 10% fetal calf serum (S0615, Biochrom) and 1% penicillin/streptomycin (P06-07050, PAN-Biotech). The confluent culture was split every 3 to 6 days. Cells were detached from the culture plate with trypsin/EDTA (PAN-Biotech) for 3 min at 37°C. Trypsin was subsequently deactivated in the solution by adding a double amount of cell culture medium. An aliquot was then transferred into a new tissue culture dish with fresh culture medium. The remaining cells could be used for experiments.