Sc 21706

Human PSCs were characterized as described previously^{84 (link)}. Briefly, the hPSCs were monitored regularly microscopically, and characterized by immunofluorescence staining (IHC) for expression of pluripotency markers using the following primary antibodies; Nanog (1:200, R&D Systems, AF1997), OCT-3/4 (1:200, R&D Systems, AF1759), SSEA-3 (1:300, Novus Biologicals NB100–1832), SSEA-4 (1:200, R&D Systems, MAB1435), TRA-1-60 (1:200, Millipore, MAB4360), TRA-1-81 (1:200, Santa Cruz Biotechnology SC-21706), and early marker for differentiation SSEA-1 (1:200, Santa Cruz Biotechnology, SC-21702). Alexa Fluor-conjugated (1:400, ThermoFisher Scientific A-11055, A-21042, A-10037), and FITC-conjugated (1:400, Novus Biologicals, NB7102) secondary antibodies were used. Nuclei were counterstained with 4′,6′diamidino-2-phenylidole (DAPI) (Vector Laboratories Inc., Burlingame, CA).
Pluripotency was verified with in vitro pluripotency assay by spontaneous differentiation as embryonic bodies^{84 (link)}, followed by immunofluorescence analysis for alpha-smooth muscle actin (SMA, 1:400, R&D Systems, MAB1420) for mesoderm, alpha-fetoprotein (AFP, 1:200, R&D Systems MAB1369) for endoderm, and OTX2 (1:200, R&D Systems, AF1979) for ectoderm. Karyotyping was performed at Finnish Microarray and Sequencing Centre (FMSC), Turku Centre for Biotechnology with the KaryoLite BoBs assay (Perkin Elmer).

Cells were harvested by centrifugation, and cell pellets were lysed with RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors. After the determination of protein concentration, we used the Western blot procedure to analyze the lysates according to previously published protocols [47 (link)]. The primary antibodies were as follows: In order to detect the MGMT protein, a polyclonal antibody from Cell Signaling Technology (Danvers, MA, USA; #2739) was used. For cleaved caspase 3, we used a monoclonal antibody (MAB10753) from Millipore-Sigma (Burlington, MA, USA). For EBNA-1, Ea-D, Zta/ZEBRA, CHOP, TRA-1-81, and beta-actin, we used monoclonal antibodies (sc-81581, sc-58121, sc-53904, sc-166682, sc-21706, and sc-47778), which were from Santa Cruz Biotechnology. As secondary antibodies, we applied horseradish peroxidase-antibody conjugates (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) according to the supplier’s recommendations. All immunoblots were repeated at least once to confirm the results. Uncropped images of Western blots along with molecular weight markers and the densitometric scanning of images are provided in the Supplementary Materials.