Fig. 4

PRX1 knockdown in vitro altered M1 morphology, cell proliferation and mesenchymal pluripotency. a In vitro culture images of M1 germs at 1, 3, 5, 7 and 9 days after transfection of Prx1 siRNA (siPrx1) or negative control (siCTRL). Scale bar: 100 μm. b Confocal images of EdU labelled proliferating dental MSCS after transfection of Prx1 siRNA (siPrx1) or negative control (siCTRL). Samples were collected 12 h after EdU administration (n = 3). Scale bar: 100 μm (c) Quantification of EdU-positive cells between siPrx1 and siCTRL group (n = 3). The error bar represents the standard deviation of the mean. ****P < 0.000 1. d Pcna mRNA expression between siPrx1 and siCTRL group was verified by RT-qPCR (n = 3). it showed significantly lower Pcna expression after Prx1 knockdown. **P < 0.01. e Flow cytometry analysis of cell cycle phases to identify the impact of Prx1 knockdown on cell cycle arrest. f mRNA expression level of mesenchymal markers Vim, N-cadherin, and Postn after Prx1 knockdown (n = 3). *P < 0.05, **P < 0.01. g Immunofluorescence for VIM (red) and cytoskeleton (Phalloidin, green) was performed to compare the mesenchymal signature and cellular shape after Prx1 knockdown to negative control. Scale bar: 25 μm. h Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed the top ten differentially expressed pathways in M1 tooth germs between siPrx1 and siCTRL group, including signaling pathways regulating pluripotency of stem cells and Wnt signaling