Zfp322a is required for ES pluripotency.

<p>(<b>A</b>) <i>Zfp322a</i> mRNA level was decreased in mES cells cultured in LIF withdrawal ESC medium. The level of the <i>Zfp322a</i> and <i>Pou5f1</i> mRNA were compared to control cells cultured in normal ESC medium and normalized against <i>β-actin</i>. (<b>B</b>) Zfp322A protein level showed a reduction as mES cells differentiated. β-actin served as loading control. (<b>C</b>) <i>Zfp322a</i> RNAi caused ES cell differentiation. AP staining was conducted on the fourth day of selection after the cells were transfected with <i>Zfp322a</i> shRNAs. <i>Zfp322a</i> RNAi cells displayed a lighter colour compared to the dark red colour of mock RNAi cells. (<b>D</b>) Pluripotency genes were down-regulated upon <i>Zfp322a</i> RNAi. ES cells transfected with empty pSUPER.puro vector were used as a control and gene expression levels were normalized against <i>β-actin</i>. (<b>E</b>) <i>Zfp322a</i> RNAi resulted in decreased Oct4, Nanog, Sox2 and Rex1 protein. β-actin served as loading control. (<b>F</b>) Quantification of the protein level changes. Protein levels of <i>Zfp322a</i> knocked-down cells were normalized against β-actin and compared to control RNAi cells using software ImageJ. (<b>G</b>) Representative flow cytometry results showed that fluorescence intensities of Oct4 and Nanog were repressed in <i>Zfp322a</i> depleted cells as compared to control cells. (<b>H</b>) <i>Zfp322a</i> depletion led to reductions of Oct4 and Nanog expression at cellular levels. The mean of fluorescence intensity was calculated using software flow software 2.5.0. Relative fluorescent intensities of <i>Zfp322a</i> RNAi cells were normalized against control knocked-down cells. Standard deviations were derived from three independent experiments. (<b>I</b>) <i>Zfp322a</i> RNAi caused up-regulation of lineage specific markers for endoderm, mesoderm and trophectoderm. Specific primers were used to check the respective gene expression levels by real-time PCR.