Alexa 647 conjugated donkey anti mouse

iPSC-BEC monolayers seeded onto 8-well ibidi μ-Slides (Ibidi, #80821) were washed with PBS and fixed with methanol or 3.7% PFA for 15 min. After washing, PFA-fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min. Washing was followed by blocking in 10% Fetal Calf Serum (Life Technologies, #10270) or 10% Goat Serum (Sigma-Aldrich, #G9023) in PBS for 1 h at RT. Primary antibodies [ZO-1 (1:100, Proteintech, #21773-1-AP), Claudin-5 (1:100, Abcam, #ab15106), Occludin (1:200, Thermo, #33-1500), and CD147 (1:100, Biorad, #MCA2882Z)] were incubated overnight at 4°C. After washing, incubation with secondary antibodies [Alexa 555-conjugated donkey anti-rabbit (Thermo, #A31572), Alexa 647-conjugated donkey anti-mouse (Thermo, #A31571) or Alexa 488-conjugated goat anti-mouse (Thermo, #A11001)] was done at room temperature for 1 h. Confocal microscopy images were acquired with a Zeiss LSM 780 microscope. ZEN software (version 2.3 blue edition) was used for image analysis.

For PTC-318, 200,000 cells were plated, treated with PTC-318, and incubated for 48 hours before collection. For shBMI1, 100,000 cells were plated and incubated for 96 hours. Doxycycline was refreshed every day. After corresponding incubation times, equal amounts of cells (500,000–2,000,000) were collected after trypsinization and thereafter centrifuged, fixed with 70% ethanol, and stored at -20°C until staining. For staining, cells were centrifuged for 3 minutes at 1,500 rpm and then washed in PBS. Cells were centrifuged again at 1,500 for 3 minutes, and pellets were resuspended in 0.25% Triton X-100 (Sigma), transferred to 1.5 ml tubes, and incubated on ice for 15 minutes for permeabilization. Cells were centrifuged at 5,000 g for three minutes, resuspended in PBS containing 1% BSA and 1:100 mouse anti-phospho-Histone H3 (Cell signaling, 9701), and incubated for 1 hour at room temperature. After washing, cells were resuspended in PBS containing 1% BSA and 1:300 Alexa 647 conjugated donkey anti-mouse (Thermofisher, A21238) and incubated for 30 min at room temperature and in the dark. Cells were thereafter washed, resuspended in 150 μl PBS containing 10 μg/ml DNase-free RNase A, and incubated for 30 min at 37°C. Prepared cells were stored at -20°C or 4°C until analysis on a flow cytometer (LSR II). Propidium iodide (1.5 μg) was added to the samples right before the assessment.