Fig 2: Sox2 induces a regeneration response in olMG cells.(A–B) Induction scheme and construct (B) used for sox2 induction. (C–C’’) Cryosection of a sox2-induced hatchling medaka retina. BrdU-positive (magenta) olMG cells, which are labeled by GS (cyan) can be detected in the INL (arrowhead). Additional BrdU-positive cells are located in the ONL, in the location of both rods and cones (open arrowheads). (D–E'') Cryosections of sox2-induced hatchling medaka retinae. BrdU-positive (magenta) ACs, which are labeled by HuC/D (cyan) can be detected in the INL (D-D'', arrowheads) and GCL (E-E'', arrowheads). Additional BrdU-positive cells are located in the ONL, in the location of both rods and cones (open arrowheads) (n = 7 sox2 OE fish and n = 8 control fish, data obtained from two independent experiments each). Scale bars are 10 µm. (F) Quantification of the location of BrdU-positive cells in sox2-induced fish (607 cells in 14 retinae) versus wild-type control fish treated with mifepristone (341 cells in eight retinae) and transgenic rx2::LexPR OP::sox2, OP::H2B-eGFP fish not treated with mifepristone (218 cells in four retinae) reveals an increase in BrdU-positive olMG cells, ACs and RGCs as well as a decrease in rod PRCs in sox2-induced fish. Wild-type control vs Sox2 OE: Rod PRC **p=0.0031, Cone PRC ns p=0.678, AC *p=0.0434, RGC *p=0.0445, MG **p=0.0083. Non-treated transgenic vs Sox2 OE: Rod PRC ***p=0.0004, Cone PRC ns p=0.528, AC *p=0.0445, RGC *p=0.0445, MG **p=0.0061. Box plots: median, 25th and 75th percentiles; whiskers show maximum and minimum data points. (G) olMG cells respond to injuries by proliferation without self-renewal and restriction toward PRC fate. Targeted expression of sox2 induces self-renewal and increased potency of olMG cells.

Fig 3: SOX2 and IGF1R as prognostic factors in bladder cancer. (a,b) Kaplan–Meier analysis of SOX2 (a) and SOX4 (b) expression with recurrence-free survival in patients from TCGA_BLCA cohort. The significance was examined by log-rank test. *P < 0.05. (c) Kaplan–Meier analysis to assess the correlation of SOX2-high/SOX4-low and SOX2-low/SOX4-high signatures with recurrence-free survival in patients from TCGA_BLCA cohort. The significance was examined by log-rank test. *P < 0.05. (d) Kaplan–Meier analysis of IGF1R expression with recurrence-free survival in patients from TCGA_BLCA cohort. The significance was examined by log-rank test. **P < 0.01. (e) Kaplan–Meier analysis to assess the correlation of SOX2-high/IGF1R-high and SOX2-low/IGF1R-low signatures with recurrence-free survival in patients from TCGA_BLCA cohort. The significance was examined by log-rank test. ***P < 0.001. (f) The schematic model of SOX2-mediated oncogenesis of bladder cancer. SOX2 induces the expression of IGF2 and IGF1R, but suppresses that of IGFBP1, thus promoting AKT phosphorylation, with increased proliferation and survival of bladder cancer cells.

Fig 4: SNEP1 is a downstream target gene of the Gli transcriptional factor.A, B Screening for novel downstream target genes of Hh signaling. Venn diagram (A) and heatmap (B) of differentially expressed genes (DEGs) (fold change =2 or = 0.05, adjusted p < 0.05) in HT-29 cells treated with GANT61 or expressing Gli2 and cluster analysis of these genes with Gene Ontology (GO) annotation. C Gli2 expression affects SNEP1 mRNA levels. HT-29 cells were transfected with Myc-vector, Myc-Gli2, or Myc-Gli2A for 48 h and harvested for qPCR analysis of SNEP1 mRNA. Bcl2, Ptch1, Sox2 and Gli1, well-known Hh signaling target genes, were used as positive controls. D Gli2 expression affects SNEP1 protein levels. HT-29 cells were transfected with Gli2 constructs for 48 h and harvested for IB with the indicated antibodies. E Gli2 knockdown reduces SNEP1 protein levels. HT-29 cells were transfected with shRNAi-Gli2 plasmids for 72 h and harvested for IB analysis with the indicated antibodies. F Pharmacological repression of Gli2 reduces SNEP1 mRNA levels. HT-29 cells were treated with GANT61 for 48 h and harvested for RNA extraction and qPCR. G Chromatin immunoprecipitation (ChIP) assays for Gli2 at the SNEP1 promoter. Upper: schematic representation of the SNEP1 promoter region showing the putative transcription factor-binding sites. Lower: HT-29 cells were harvested for ChIP assays as described in the Materials and Methods with IgG or anti-Gli2 antibodies for IP followed by qPCR with specific primers for each putative-binding element as indicated. H Luciferase assays for Gli2 transcriptional activity at the SNEP1 promoter. A series of SNEP1-luciferase constructs (left) were transfected into HEK-293T cells, and relative Gli2 transcript levels were measured 48 h after transfection (right). Each experiment was performed in triplicate. *p < 0.05, **p < 0.01.

Fig 5: SOX2 promotes migration of endometrial carcinoma cells. (A) qPCR analysis to measure SOX2 (left) and EGFR (right) expression in Ishikawa-02 (Ish-02) and Ishikawa-02-S (02-S) cells. Ishikawa-02-S is a stable clone selected from 02/D10, the pooled Ishikawa-02 cells previously treated with TGF-ß (1 ng/ml) for 10 days. ***P < 0.001. (B) A clonogenic analysis to compare the growth of Ishikawa-02 (Ish-02) and Ishikawa-02-S (02-S) for 14 days. Colonies were subjected to crystal violet staining (upper) and quantified by ImageJ analysis (lower). ***P < 0.001. (C) Cell tracking analysis to monitor the trajectory (upper) and quantify migrated distance (lower) of Ishikawa-02 (Ish-02) and Ishikawa-02-S (02-S) cells under a normal serum condition. ***P < 0.001, n = 10 cells/group. (D) Cell tracking analysis to monitor the trajectory (upper) and quantify migrated distance (lower) of Ishikawa-02 cells treated with EGF (100 ng/ml) (EGF) for 24 hr under a serum starvation condition. ***P < 0.001, n = 10 cells/group.