Fig. 2

Stable shRNA knockdown of Oct4A in epithelial ovarian cancer cell line HEY. a Four established epithelial ovarian cancer cell lines OVCAR5, SKOV3 OVCAR433 and HEY were screened for endogenous expression of Oct4A mRNA by qPCR analysis. Relative quantification of Oct4A mRNA expression was standardized to 18S housekeeping gene and normalized to the normal ovarian epithelium immortalized cell line ISOE398. b shRNA-mediated silencing of Oct4A in HEY cells (Oct4A KD1 and Oct4A KD2) was confirmed by qPCR analysis. Relative Oct4A levels were normalized to 18S housekeeping gene and calibrated to vector control samples prepared in triplicate. c Western blot analysis using nuclear cell lysates and 12 % SDS-PAGE gels further validating Oct4A knockdown in Oct4A KD cells as determined by protein bands present at ~50 kDa. Total protein load was determined by stripping and re-probing the membrane with GAPDH. d Oct4A protein expression was significantly reduced in Oct4A KD cells as determined by densitometry analysis. Data is expressed as a ratio of Oct4A protein expression compared to GAPDH protein expression. e Phase contrast images of exponentially growing HEY cells cultured on plastic indicating no obvious morphological changes following shRNA-mediated knockdown of Oct4A. Magnification is set at 100x. Scale bar represents 50 μM. f-g Lin28 and Sox2 mRNA expression was investigated by qPCR analysis. Relative Lin28 and Sox2 levels were normalized to 18S housekeeping gene and calibrated to vector control samples prepared in triplicate. h-i Lin28 and Sox2 protein expression in Oct4A KD cells was investigated by Western blot analysis. Cytoplasmic cell lysates were prepared for Lin28 analysis and nuclear cell lysates used for Sox2 analysis. Total protein load was determined by stripping and re-probing the membrane with GAPDH. For all graphs sets, data is expressed as the mean fold change ± SEM from three independent samples. Significance is indicated by *p < 0.05, **p < 0.01 and ***p < 0.001 and determined by One-Way ANOVA using Dunnett's post-test compared to the ISOE398 cell line or HEY vector control