Cdk2ap1

Under different experimental conditions, hESCs were seeded onto four chambered glass slides. Paraformaldehyde (PFA, 4%) in PBS was used for fixation, permeabilization for intracellular markers was achieved with 0.2% Triton X-100 in PBS and blocked with normal goat serum. Fixed cells were incubated with primary antibodies: CDK2AP1 (Santa Cruz, CA, USA) and Anti-Phospho-Histone-3 (Cell Signaling, MA, USA). Goat anti-rabbit IgG conjugated to Alexa 594 (Invitrogen, CA,) was used as a secondary antibody. Fluorescent images were acquired using a Cool- Snap EZ camera (Photometrics, Tucson, AZ) mounted on a Nikon Eclipse TE 2000-S inverted microscope (Nikon, Melville, NY) with attached image analysis software. All image settings were controlled for uniform acquisition between samples. Specifically, uniform exposure time was maintained for images acquired from experimental samples as well as negative controls for background subtraction. For experiments involving determination of mitotic index based on phospho-histone 3 staining, random fields were selected and counting was performed in triplicate by counting around 500 cells during each analyses. Mitotic index was calculated by dividing the number of phospho-histone 3 positive cells, over the total number of cells.

Western Blot was carried out as previously described [12 (link)]. Cells for analysis were harvested by trypsinization, centrifuged at 1000 rpm and washed once with ice-cold PBS buffer. Cells were then lysed using the Total Protein Extraction Kit (EMD Millipore, MA)) based on protocol provided by the manufacturer. Cell lysates were then subjected to Western blot analyses using specific antibodies to various cyclins. Cell lysates were prepared from wild type and CDK2AP1 knockdown WA09 hESCs and analyzed for CDK2AP1, OCT4, NANOG, p53, Cyclin A1 expression by Western blot using specific antibodies. The CDK2AP1, OCT4, NANOG and Cyclin A1 antibodies were obtained from Santa Cruz, CA, USA, while p53 antibody was obtained from Cell Signaling, MA, USA. Details of antibodies and sources are provided in S2 Table. Appropriate infrared emitting-conjugated secondary antibodies were obtained from Invitrogen, CA. Detection was then carried out using the Odyssey Infrared Imaging System (Li-Cor Biosciences, NE). The quantitative analysis of the Western Blot bands was carried out using the ImageJ software [13 (link)].