Fig 2: SOX2 induces IGF2/IGF1R signaling molecules in bladder cancer cells. (a) qPCR analysis to assess IGF2 expression in T24 cells transduced with the lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector (Ctrl). Results are the average of three replicates and expressed as the mean ± S.D. ***P < 0.001. (b) qPCR analysis to assess IGF2 expression in T24 versus 5637 cells (left) and in 5637 cells transduced with shSOX2 versus scrambled control (SC) vector (right). The #1 and #2 indicate the two distinct shRNAs that target different regions within SOX2. ***P < 0.001. (c) RNA-seq analysis of SOX2 and IGF2 expression in ESC (HUES3) and differentiated fibroblast cells from GSE73211 database. (d) ChIP-qPCR analysis to assess H3K4me3 levels of IGF2 region in SOX2-expressing T24 cell (left) and SOX2-silenced 5637 cells (right). **P < 0.01, ***P < 0.001. Primers targeting the H3K4me3 signal of IGF2 were designed at chr11: 2,158,962–2,159,038. (e) qPCR and immunoblotting analysis of IGFBP1 expression in SOX2-overexpressing T24 cells (left) and SOX2-silenced 5637 cells (right). Results are the average of three replicates and expressed as the mean ± S.D. *P < 0.05, ***P < 0.001.

Fig 3: SOX2-induced IGF2/IGF1R signaling is essential for bladder cancer cells. (a) qPCR (upper) and immunoblotting (lower) analysis of 5637 cells transduced with the lentiviral vector encoding shRNA against IGF2 (shIGF2) or scrambled control vector (SC). The #1 and #2 indicate the two distinct shRNAs that target different regions within IGF2. ***P < 0.001. (b) Clonogenic assay of 5637 cells transduced with shIGF2 or scrambled control vector (SC). Colonies were subjected to crystal violet staining (top) and quantified by ImageJ analysis. Results are the average of three replicates and expressed as the mean ± S.D. **P < 0.01. (c) qPCR (upper) and immunoblotting (lower) analysis of 5637 cells transduced with the lentiviral vector encoding shRNA against IGF1R (shIGF1R) or scrambled control vector (SC). The #1 and #2 indicate the two distinct shRNAs that target different regions within IGF1R. ***P < 0.001. (d) Clonogenic assay of 5637 cells transduced with shIGF1R or scrambled control vector (SC). Colonies were subjected to crystal violet staining (top) and quantified by ImageJ analysis. Results are the average of three replicates and expressed as the mean ± S.D. **P < 0.01. (e) Immunoblotting analysis to assess the expression of phosphorylated AKT at Ser473 and total AKT expression in SOX2-expressing T24 cells under low-serum (1% FBS) condition in the presence or absence of linsitinib (5 µM) for 48 hr. (f) 3D colony-forming analysis to assess linsitinib effect on colony formation of SOX2-expressing T24. The cells were grown under the low-attached condition in low-serum medium (1% FBS) in the presence or absence of linsitinib (5 µM) for 7 days. Photos (left) are representative images of the colony and colony sizes were quantified (right). Results are the average of three independent experiments and expressed as the mean ± SD. ***P < 0.001.