Fig 2: Exosomal miR-146a directly targets Numb in ß-cells. a Immunofluorescent staining for NUMB expression in pancreatic sections (scale bar 20 µm). RT-qPCR analyses of the mRNA levels of Numb in islets transfected with miR-146a mimics (b) or treated with an miR-146a inhibitor combined with bmMDEs (d). Western blotting for NUMB in INS-1 cells transfected with miR-146a mimics (c) or treated with an miR-146a inhibitor combined with bmMDEs (e), ß-actin was used as the loading control. f Schematic of the sequence that miR-146a targets in the WT or mutated 3'UTR of Numb mRNA. g Luciferase activity of Numb WT or mutated 3'UTR reporter plasmids in INS-1 cells transfected with miR-146a mimics or miR-NC mimics as indicated. A GSIS assay was performed to detect insulin stimulation in islets transfected with Numb overexpression (h) or siRNA (m). RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets transfected with Numb overexpression (i–l) or siRNA (n–q). Data are presented as the mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig 3: Overexpression of Oct4 enhances Egr1 expression, OPN production, and migratory capability of human lung cancer cells. a and b, H1299 cells (left panel) were transiently transfected with 6 µg of pSin-EF2-Oct4-Pur or the control plasmid pSin-EF2-Pur, and their RNA (a) and protein (b) levels of Oct4 and Egr1 were analyzed at 48 post-transfection by RT-PCR (a) and immunoblot (b) analysis. A549 cells (right panel) stably overexpressing Oct4 and vector control cells were examined for Oct4 and Egr1 expression by RT-PCR (a) and immunoblot (b) analysis. Expression of GAPDH (a) and ß-actin (b) served as the loading control. c, The levels of OPN in the supernatants of Oct4-overexpressing and control A549 cells were quantified by ELISA. d, Migratory capabilities of A549-Oct4 and A549-vector cells were detected by a Boyden chamber assay. Cells that migrated through the membrane of the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy and photographed (left panel), and three fields in each membrane were counted (right panel). Data are mean ± SEM. **, p < 0.01

Fig 4: Oct4, Egr1, and OPN are overexpressed in lung cancer and Egr1 expression is correlated with shorter survival. a, Representative staining for Oct4, Egr1, and OPN in human lung tumors graded as low and high expression. b, Numbers of tumors expressing high or low levels of Egr1 in tumors with high or low Oct4 expression. c. Numbers of tumors expressing high or low levels of OPN in tumors with high or low Egr1 expression. d, Tumor recurrence rates between high- and low- expression of Oct4, Egr1, and OPN in lung cancer patients, none of them reached statistical significance (Oct4: p = 0.273; Egr1: 0.261; OPN: 0.253). e-g, Kaplan-Meier curves for disease-free survivals in patients with high (red line) and low (black line) Oct4 (e), Egr1 (f), and OPN (g) expression

Fig 5: Germ layer and neural differentiation showed similar cell fate between WT and Fahd1-KO iPSCs. (A) Phase contrast imaging showing typical iPSC-like round colonies when cultured on inactivated MEFs. (B) Immunofluorescence showing the expression of the pluripotency markers Oct4 and Sox2 in both WT and KO iPSCs. Cell nuclei were stained with DAPI. (C) After germ layer differentiation, immunofluorescence on the differentiated cells showed the expression of the endodermal marker AFP, the mesodermal marker SMA, and the ectodermal marker Tuj1. (D) Neural differentiation showed typical neural rosette formation both in WT and Fahd1-KO, as it is shown in the phase contrast images. (E) Relative mRNA expression by qRT-PCR of the pluripotency marker Nanog, and the neural progenitor markers Pax6 and Nestin in WT and Fahd1-KO MEFs, iPSCs, and neural progenitors. The results showed a downregulation of the pluripotency and an upregulation of neural markers similarly in both WT and Fahd1-KO. MEFs: mouse embryonic fibroblasts, iPSCs: induced pluripotent stem cells, WT: wild type, KO: knock-out, ND: not detected. Scale bars, 100 µm.