Fig 2: Isolation of inner cell mass and derivation/characterization of embryonic stem cell lines. a Isolated ICM and trophoblast from rhinoceros embryo after mechanical dissection with insulin needles (40×); b ICM outgrowth 48 h after plating on feeder cells (20×). c Representative image of ES colonies 24 h after enzymatic passaging; d ES colonies ready for the next passaging. Characterization of undifferentiated ES colonies: e, i, o OCT4 staining; f, l, p NANOG staining; g, m, q SOX2 staining; h, n, r SSEA3 staining. Characterization of differentiated derivatives: s p75 staining of neural crest precursors; t, u Tuj1 and TH staining of neuronal cells; v, z, x GATA4, GATA6, SOX17 staining of endodermal cells; y, w MYOSIN, NKH2.5, TNNT2 staining of myocardial cells. Bar 100 µm; j RT-PCR for SOX2, OCT4, and NANOG in fibroblasts and rESC (n = 1 sample for each cell line)

Fig 3: Src inhibition effects and SDCBP overexpression. (A) The indicated cell lines were transduced with a siRNA against Src (siSrc) or a scramble siRNA (siCtrl) and examined for Src inhibition at 24 and 48 h post-transfection. Quantification of p-Src/Vinculin, total Src/Vinculin (p-Src/Src ratio is not indicated), SDCBP/Vinculin, CD44/Vinculin, and Sox2/Vinculin expression is indicated. Note the decrease in the Src total levels and p-Src levels in CCL-138-R cells. Note also the decrease in the CD44 protein in all cases; (B) Survival curves of cells depleted of Src in response to CDDP. Note that Src inhibition sensitizes HTB-43-R and CCL-138-R cells to CDDP; (C) Flow cytometry study indicating HTB-43 cells positive for ABCB1 upon Src inhibition (upper panel). The percentages of each sample and the reduction are indicated in the table (lower panel); (D) The indicated cell lines were transduced with pcDNA-SDCBP, and SDCBP RNA expression was evaluated by qRT-PCR at 24 h post-transfection; (E) Western blot and quantification of the SDCBP/Vinculin, p-Src/Src ratio, CD44/Vinculin, and Sox2/Vinculin levels in cells from the experiment shown in Figure 4D; (F) HTB-43 and CCL-138 cells overexpressing SDCBP were treated with CDDP, and their response was determined via IC50 values. (G) Table representing the IC50 values of each condition of the experiment shown in Figure 4F and the percentage of increase; (H) Graphs indicating the number of spheres generated from cells overexpressing SDCBP; (I) Analysis of ABCB1 expression by flow cytometry of HTB-43 cells overexpressing SDCBP. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: A proposed model for the involvement of CEBPD in stemness and TMZ resistance in GBM.CEBPD can facilitate GSC formation and GBM resistance to TMZ by upregulating the SOX2, OCT4, NANOG, and ABCA1 expression through acquired and intrinsic regulation.

Fig 5: Immunohistological analysis of the telencephalon of Tg rat embryo. (A,B) Low (A) and high (B) magnification of coronal sections of the telencephalon demonstrating immunoreactivity of SOX2 and GFP fluorescence. (B) Tg rat telencephalon images showed GFP signals (green) not only around SOX2-positive cells (red) of the VZ but also in SOX2-negative cells (blue), as shown in the magnified view (white boxes). No GFP signals was observed in the Wt telencephalon. Scale bars: 100 µm.