Effect of enzyme digestion into cell clumps on protein levels of OCT4 and SOX2 in human embryonic stem cells.

Objective. To reveal the effect of enzyme digestion into cell clumps on protein levels of octamer-binding protein 4 (OCT4) and sex determining region Y-box 2 (SOX2) in the human embryonic stem cells (hESCs) and its possible molecular mechanism. Methods. 1 Western blotting was conducted to detect the protein levels of OCT4 and SOX2 in the hESCs treated by enzyme digestion into cell clumps [Dispase or collagenase type IV (COL4)], enzyme digestion into single cells (Accutase or trypsin), or mechanical scraping dissociation. 2 Ethylene glycol tetraacetic acid (EGTA) was used to destruct hESC-hESC interaction, and protein levels of OCT4 and SOX2 in the hESCs treated with EGTA alone or combined with COL4 were detected by Western blotting. 3 Quantitative proteomics analysis based on mass spectrometry was carried out to identify differentially expressed total proteins and phosphorylated proteins in the hESCs treated with COL4 alone or in combination with EGTA. 4 hESCs were pre-treated with proteasome inhibitor (bortezomib) and lysosome inhibitor (chloroquine), respectively, followed by Western blotting and immunofluorescence staining to measure their effects on COL4-induced down regulation of OCT4 and SOX2. 5 Western blotting analysis was conducted to test the effect of COL4 treatment on cofilin as well as Rac1 inhibitors (EHT1864 and EHop-016) treatment on the protein level of SOX2 in the hESCs. Results. 6 COL4 or Dispase treatment led to protein levels decrease of OCT4 and SOX2, while Accutase, trypsin and mechanical dissociation did not elicit such effect. 7 EGTA rescued OCT4 and SOX2 protein levels decrease indcued by COL4 treatment. 8 Quantitative mass spectrometry and functional enrichment analysis suggested that microtubule, actin filament (F-actin) and lysosome-associated proteins might be related to OCT4 and SOX2 protein levels decline in the suspended clumps of hESCs upon COL4 treatment. 9 Chloroquine, instead of bortezomib, could block COL4-induced protein levels decrease of OCT4 and SOX2. 10 The treatment of COL4 or Rac1 inhibitors could downregulate protein levels of cofilin and SOX2. Conclusion. Upon hESC detachment in cell clumps induced by enzyme treatment, the protein levels of SOX2 and OCT4 decline, which may be related to Rac1/cofilin/F-actin signaling pathway and lysosome. [ABSTRACT FROM AUTHOR]