Fig. 3

Suppression of Oct4A resulted in reduced spheroid forming ability and a loss of Lin28 and Sox2 in HEY cells. a The spheroid forming ability of HEY cells following stable shRNA-mediated knockdown of Oct4A was assessed by culturing cells on ultra-low attachment plates as described in the Materials and Methods. Cellular aggregation was monitored over 18 days and documented using phase contrast microscopy. Images are representative of three independent experiments set at 100x magnification. Scale bar represents 100 μM. b The number of spheroids produced after 18 days was assessed using a phase contrast microscope calibrated with an ocular micrometer. Spheroids with a diameter greater than 200 μM were classified as spheroids. c Expression of Oct4A, Lin28 and Sox2 in Oct4A KD 18 day spheroids was evaluated by qPCR analysis. Relative quantification of Oct4A, Lin28 and Sox2 mRNA expression in Oct4A knockdown cells was standardized to 18S housekeeping gene and normalized to vector control cells