Plasmids and transfections

The overexpression constructs of Oct4 (pcDNA 3.3 OCT4, plasmid no. 26816), Sox2 (pSin-EF2-Sox2-Pur, plasmid no. 16577), HDAC1 (HDAC1 Flag, plasmid no. 13820), and the shRNAs against Oct4 (LL-HOCT4i, plasmid no. 12198), and Sox2 (pLKO.1 Sox2 3H b, plasmid no. 26352) were purchased from Addgene. The ABCG2 siRNA (NM_004827), HDAC1 siRNA (NM_004964), and HDAC2 siRNA (NM_001527) were purchased from Sigma-Aldrich. The SMAR1 overexpression plasmid (SMAR1 cDNA) and SMAR1 shRNA were gifts from S. Chattoadhyay (10, 14). The SMAR1 overexpression plasmid produces a GFP-tagged SMAR1 protein that gives a separate band of external SMAR1-GFP when visualized in Western blot (10).

The plasmids and control vectors were introduced into cells and spheres using Lipofectamine-2000 (Invitrogen) according to the protocol provided by the manufacturer. Stably expressing clones were isolated by limiting dilution and selection with puromycin dihydrochloride (1 μg/ml; Sigma-Aldrich) or G418 Sulfate (1 mg/ml; Sigma-Aldrich), and the resistant cells were cloned and screened by Western blotting with specific antibodies. The siRNA were transfected using Lipofectamine-2000, and cells were harvested after 34 to 36 hours after which the knockdown efficiency was determined using Western blot.