Dylight488 maleimide

For binding studies PMT was labelled covalently with DyLight488 maleimide (Thermo Fisher Scientific) according to the manufacturer‘s instructions. Pre-cultured HeLa, HepG2 or MEF cells and derivates were detached from culture plates using 10 mM EDTA in PBS. For each condition 2 × 105–2.5 × 105 cells were used and incubated for 20 min with the indicated PMT-DyLight488 concentrations at 4°C. Following several washing steps with ice-cold TBS, cell bound fluorescence was measured using a FACS Melody (BD Bioscience). Via the appropriate Software FACSChorus (BD Bioscience) 10.000 cells were analyzed. After gating and the average of surface bound fluorescence of all cells was given.
For analysis of surface bound fluorescence of cells, the median forward scatter of the analyzed Hela and HepG2 cell populations was measured for normalization.

HA (M_w = 20 kDa) was a product of Lifecore Biomedical (Chaska, MN, USA). Gilteritinib (GLT) was acquired from MedChemExpress (South Brunswick, NJ, USA). EGCG was a product of DSM Nutritional Products Ltd. (Heerlen, The Netherlands). Amicon Ultra-15 centrifugal filter devices, NaCl, and urea were obtained from Merck Millipore Corporation (Darmstadt, Germany). Triton X-100, Tween 20, bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI), and dextran from Leuconostoc spp. (M_w = 15–25 kDa) were obtained from Sigma-Aldrich (St. Loius, MN, USA). RPMI 1640 media, DMEM media, penicillin/streptomycin solution, fetal bovine serum (FBS), phosphate-buffered saline (10 mM, pH 7.4, PBS), DyLight 488 maleimide, CellROX Green oxidative stress detection reagent, and Image-iT LIVE Red apoptosis detection kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Caspase-Glo 3/7 assay kit and CellTiter-Glo viability assay reagent were obtained from Promega (Madison, WI, USA). Glass-bottom microwell dishes (dish size = 35 mm, thickness = 0.13–0.16 mm) were obtained from MatTek Corporation (Ashland, OR, USA). Float-A-Lyzer tubing (M_w cutoff = 3.5–5 kDa) was a product of Spectrum Laboratories (Piscataway, NJ, USA).

Abberior STAR 635P maleimide was purchased from Abberior (Germany). Alexa Fluor 488 C5 maleimide, Alexa Fluor 568 C5 maleimide, Alexa Fluor 594 C5 maleimide, Alexa Fluor 647 C2 maleimide, DyLight405 maleimide, DyLight488 maleimide, Oregon Green 488 maleimide, and SYPRO orange were purchased from ThermoFisher Scientific (Germany). Atto488, Atto647N, and Atto565 maleimides were purchased from ATTO-TEC (Germany). Cy3, Sulfo-Cy3, Cy5, and Sulfo-Cy5 maleimides were purchased from Lumiprobe (Germany). All maleimides were incubated with a 10× excess molar ratio of l-cysteine for 1 h for quenching before the FG phase permeation assays. DAPI, Hoechst 33342, Hoechst 33258, Hoechst 34580, Oxazole yellow, Ethidium bromide, Fluorescein, Rhodamine B, and Thioflavin-T (ThT) were purchased from Sigma-Aldrich (Germany). Nuclear yellow was purchased from Abcam (Netherlands).