Fig 2: 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 3: 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.

Fig 4: MSI1-mediated mRNA interaction promotes ICAM1 translation. Representative immunoblotting analysis of ICAM1, Flag, and LIN28 in (A) U87MG cells. ß-Actin was used as the loading control (upper panel). RNA-immunoprecipitation of Flag-control or Flag-MSI1-overexpressing cell lines was performed using an antibody against Flag (IP-Flag). RT-PCR analysis for detecting mRNA expression of ICAM1, p21, and GAPDH (lower panel). To confirm RNA-mediated inhibition of MSI1, RNase A was used to eliminate total RNA in vitro, demonstrating that MSI1 promotes ICAM1 translation in an RNA-dependent manner. Input was 5% of total mRNA, and IgG was used as a negative control. (B) Identification of the effect of MSI1 on stabilizing ICAM1 mRNA in the U87MG cell line. Control or MSI1-overexpressing U87MG cells were treated with the transcriptional inhibitor actinomycin D (Act D, 6.5 µg/ml), and total RNA was harvested at 0, 1, 2 3 and 4 hr. posttreatment. ICAM1 mRNA levels were determined relative to GAPDH by RT-PCR and quantitated using ImageJ software.

Fig 5: 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).