Fig 2: Pluripotent stem cell marker protein expression. (A) Immunocytochemistry for SSEA1, SSEA3, and SSEA4. Merged representative images of SSEAs (red) with DAPI (blue) are shown. Confocal laser microscopy was used to detect SSEA1 and ZsGreen fluorescent signal. SSEA1 was positive in Cl1 and Cl3 but not in Cl2 nuclear transfer-drived ESCs (ntESCs) nor in the parental pluripotent stem cells (piPSCs). Note that ZsGreen was negative in the Cl1 cells. SSEA3 and SSEA4 were positive in all cell lines. Scale bars: 100 µm. (B) Detection of six pluripotent stem cell marker proteins using western blotting. All marker proteins were detected in the established clones. a-tubulin control. (C) Immunocytochemistry for six pluripotent stem cell markers. OCT3/4, SOX2, KLF4, c-MYC, and NANOG proteins were detected in the nucleus, while LIN28A was cytoplasmic. Although only representative images for Cl1 are shown, similar results were confirmed in Cl2 and Cl3. Scale bars: 50 µm.

Fig 3: ICAM1 is a downstream target of MSI1-modulated cell invasion. (A) ICAM1 is the most highly expressed protein in comparisons of U87/MSI1 with U87/Ctrl GBM cells. (B) Left: Expression of MSI1, ICAM1, and LIN28 in GBM-U87MG/Ctrl, and GBM-U87MG/MSI1. Right: Expression of MSI1, ICAM1, and LIN28 in GBM-05MG/sh-Scr and GBM-05MG/sh-MSI1 cells. ß-Actin was used as a loading control. (C) Left: Transwell invasion assay in GBM-05MG cells transfected with sh-Scr, sh-MSI1 or sh-MSI1/ICAM1. All data are presented as the mean ± SD, n = 10, *P < .01 by Student's t-test. Right: Transwell invasion assay in GBM-U87MG cells transfected with Ctrl, MSI1 and MSI1/sh-ICAM1. All data are presented as the mean ± SD, n = 10, *P < .01 by Student's t-test.

Fig 4: The suppression of miR-17/92 cluster by Fhit-Lin28 axis. (A and B) The regulation of miR-20a and -20b by Fhit gene. qRT-PCR analysis showing the suppressed expression of miR-20a and -20b miRNAs in Fhit-deficient HEK293 and A549 cells (A) whereas enhanced expression in A549 expressing Fhit cDNA. (C and D) Analysis of cell lines expressing Lin28. The indicated cells were harvested and prepared for qRT-PCR (C) or Western blot analysis (D). The results were confirmed in an independent biological replicated experiment. (E) Suppressed miR-20a and -20b in response to Lin28b deficiency. The indicated cell lines were transiently transfected by siLin28 siRNAs for 48 hours. Subsequently total RNA was extracted and subject to qRT-PCR analysis. The knock-down efficiency was also shown at left panel of each group of data. Three independent experiments were performed in triplicate (n=3). Bars mean ±S.D. and the p-value were obtained by student t-test (*p<0.05; **p<0.01).

Fig 5: The expression of stem cell factors in Ad-MSCs varies depending on the donor site. The expression levels of the stem cell factors (a) Oct4a, (b) Lin28A, (c) Nanog, and (d) Sox2 were determined by semi-quantitative RT-PCR in Ad-MSCs derived from adipose tissue of the knee, limb, abdomen, thigh, and hip (N = 4 each). The PCR products were loaded twice (n = 2) to minimize the loading errors. The signal intensities were quantified with the help of the ImageJ software. (e) Representative images for the RT-PCR products from the pooled samples; (f) Representative images for the Western blot signals obtained from the pooled samples; * p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated.