Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1
Beztsinna N.1, de Matos M.B.C.1,2, Walther J.1, Heyder C.2, Hildebrandt, E.2,3,Leneweit G.2, Mastrobattista E.1, Kok R.J.1*1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands2ABNOBA GmbH, Pforzheim, Germany3Institute for Mechanical Engineering and Mechanics, Karlsruhe Institute of Technology, Karlsruhe, Germany*corresponding author, email@example.comFigure S1 A. FPLC elution profile for ML1 (0.6M NaCl gradient). The main peak represents purified ML1 cytotoxin, with a small left shouldercomprising the variant (A1) of the apoptotic A chain typical for winter mistletoe harvest. B. SDS-PAGEanalysis of ML1 under non-reducing and reducing conditions. Monomer ML-1 and multimers of ML-1are observed under non-reducingconditions. Disruption of disulfide bonds under reducing SDS-PAGE conditions separates the covalently linked A and B chain of ML-1 into 26 kDa and 30 kDa fragments, respectively. A third band was detected at 29 kDa, which is a variant (A1) of the A chain found in winter harvests of the mistletoe plant.
Figure S4. Confocal imagesof CT26 cells pre-incubated with uptake inhibitors and then incubated with ML1 for 2h (10 μg/mL) (up); Average ML1 fluorescence (n=4) in the cells quantified with Columbus suite software and normalized by subtraction of baseline cell fluorescence; nuclei are stained in blue, ML1 –red; size bar 50 μm.
Figure S5. Confocal images of CT26 cells pre-incubated with uptake inhibitors and then incubated with ML1 for 1h (10 μg/mL) (up); Average number of spots per cell (n=3) quantified with Columbus suite software; nuclei are stained in blue, ML1 –red; size bar 20 μm.
Figure S6. Confocal images of ML1 uptake (10 μg/mL, 2h) and co-staining with endo-lysosomal pathway specific antibodies; nuclei are stained in blue, ML1 –red, secondary antibody (Anti-Goat conjugated to Alexa Fluor 488) -green; size bar 50 μm.
Figure S7. A. Confocal images of ML1 uptake in 4T1 control and doxorubicinresistantcellsat 30 min incubation with 10 μg/mL ML1; nuclei are stained in blue, ML1 –red;size bar 20 μm.;
B.Pearson correlation coefficient dynamics of co-localization ML1 withendosomes,lysosomes and Golgi in 4T1 motherand resistant cells.Supplementary movies:1 and 2: Time-lapse movies of ML1 induced apoptosis in CT26 cells (1) or untreated CT26 cells in the same experiment (2); nuclei are stained in blue, apoptotic cells in green (CellEvent® live staining), ML1 in red, magnification 60x.The first 8 frames of each movie are taken every 15 min for 2h and the rest 24 frames every 3 hours up to 72h.3 and 4: Time-lapse movies of ML1 induced apoptosis in4T1 Res cells (3) or untreated 4T1 Res cells in the same experiment (2); nuclei are stained in blue, apoptotic cells in green (CellEvent® live staining), ML1 in red, magnification 40x. In total18framesare takenwithimaging every 4hours up to 72h.
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