Fig 2: SOX2 inhibition suppressed Ewing’s sarcoma growth in vivo. a Tumor growth was significantly repressed by siSOX2 intratumoral injection as shown in the curve graph (Data are presented as the mean ± S.D. **P < 0.001). b SOX2 inhibition substantially decreased xenograft tumor volume, shown as representative images exhibited. c Representative images of IHC analysis demonstrated the significant repression of SOX2, p-Akt and cyclin-E, and the enhancement of cleaved-caspase-3 in tumor samples treated with siSOX2. d Variations of the upstream regulators, such as SOX2, PI3K, Akt and p-Akt, the downstream anti-survival of proteins, such as Fas, caspase-8, Bad, caspase-9, caspase-3, p21 and p27, and the pro-proliferation factors, such as PARP and cyclin-E, were all followed the same trends described as in vitro, as analyzed by WB. The data are representative of three independent experiments

Fig 3: HepG2 cell growth in CAM tissues. (a) Representative images of H&E staining, Ki-67 and p27 immunohistochemistry, and TUNEL assay results. The magnification of the objective lens was 20-fold, and that of the ocular lens was 10-fold, such that the total magnification was 200-fold. (b) A massive reduction in the number of Ki-67-positive cells was observed after incubation with I-ol at the lowest concentration. (c) Induction of p27 protein expression. (d) Linear increase of apoptotic cells, compared with the control groups. The results are shown as the mean ± SD. Statistical calculations were performed based on two-way mixed ANOVA, followed by Bonferroni's post hoc test. In all cases, the significance level was ∗p < 0.05 (significant), ∗∗p < 0.01 (highly significant), or ∗∗p < 0.001 (very highly significant). Dose-dependent effects of I-ol were calculated by determining Pearson's correlation coefficient (r) and performing linear-regression analysis (adjusted R2), with the following statistical parameters: +p < 0.05 (significant), ++p < 0.01 (highly significant), and +++p < 0.001 (very highly significant).

Fig 4: SOX2 inhibition induced G1/S cell cycle arrest. a, and b The cell cycle was assayed by flow cytometry. The percentages of cell cycle phases were displayed in bar charts. siSOX2 transfection resulted in a significant increase of the cells proportion in the G0/G1 phases of A673 and RD-ES cells. c SOX2 inhibition amplified the expression of p21 and p27, and repressed cyclin-E, but had no effect on cyclin-D expression in Ewing’s sarcoma cells according to assayed by WB analyses. The data are representative and selected from one of three independent experiments

Fig 5: mRNA-expression levels of genes related to apoptosis and cell-cycle progression in HepG2 cells. (a, b) Cutoff values of =+5 and =-7.5 were determined, which resulted in the selection of 21 out of 88 analysed genes. These most dysregulated genes associated with cell-cycle progression and apoptosis were selected for further validation. BIRC5 expression was included in both arrays. Genes whose relative mRNA-expression levels could not be evaluated (due to no detectable amplification) were excluded from the analysis. (c) The relative mRNA-expression levels of the selected 21 genes were analysed in three independent experiments performed with HepG2 cells treated with I-ol (100 µM) for 48 h. The expression level of each gene was ranked, where the highest expression levels corresponded with the highest rank. Finally, the sums of all three ranking values were calculated for each gene and are depicted as stacked columns. In concrete terms, this ranking indicated that TNFSF11 was the least expressed gene and that TNFSF15 the most expressed gene. Three differentially expressed genes (TNFSF15, CDKN1B, and CDKN2B) showed repressed mRNA expression compared to the control group treated with 100 µM CD.