Fig. 2

Platr10 is required for the maintenance of pluripotency. A Reactivation of Platr10 in reprogramming. Skin fibroblasts were reprogrammed using lentiviral Oct4-Sox2-Kilf4-c-Myc (OSKM). Cells were collected at different stages of reprogramming and the expression of Platr10 was measured by RT-PCR. FIB, fibroblasts; iPSC, induced pluripotent stem cells; non-iPCS (un-reprogrammed cells), cells that express the four viral OSKM factors, but fail to complete reprogramming. β-Actin was used as the PCR control. Throughout the manuscript, the data are presented as the mean ± SD from three independent experiments unless they are specifically defined. **p < 0.01 as compared with FIB and non-iPSCs. B Platr10 expression is associated with Sox2 and Oct4 expression during embryoid body (EB) differentiation. iPSCs were collected at different stages of EB formation for quantitative PCR. C Requirement for Platr10 in the maintenance of pluripotency. shPlatr10-1, shRNA vector that targets Platr10 lncRNA; shCT, random shRNA control; Vector, lentiviral vector control. Platr10 was knocked down by shRNA lentiviruses in E14 cells. Cells transfected with lentiviruses carrying a random shRNA (gCT) were used as the control. The lentivirus-transfected cells were tracked by the co-expressed copGFP. Pluripotency status was examined by immunohistochemical (IHC) staining of stem cell marker NANOG. Note that the exit of iPSCs from pluripotency in the shRNA-copGFP expressing cells is accompanied by altered cell morphology and the loss of NANOG protein (red arrow). The cell islands that escape lentiviral shPlatr10 transfection are marked by a yellow dotted line. These cells maintain the same stem cell pluripotency as the iPSCs. C Platr10 is essential for optimal activity of core stem cell factor genes in iPSCs. After lentiviral transfection, iPSCs were selected by puromycin. The mixed stable cells were collected for qPCR quantitation. **p < 0.01 as compared with the Vector and shCT controls. D Platr10 enhances cell reprogramming. MEF cells were transfected with the lentiviruses carrying Platr10, the empty vector (Vector), and CTL (lncRNA control containing Platr10 antisense RNA). After doxycycline (DOX) induction, iPSC colonies were detected using an alkaline phosphatase (AP) staining kit and were quantitated as iPSC colonies per microscope field. **p < 0.01 as compared with the Vector and CTL controls