Fig 1: Aberrant expression of SOX genes in PTHS cells and organoids.a Ratio of TPM abundances for SOX genes in NPCs in 2D culture. N = 4 pairs (symbols). SOX gene subfamilies are shown above. b Top: SOX3 TPM expression in NPCs. Bottom: SOX3 relative expression (RT-qPCR). N = 4 pairs (symbols). c SOX3 is downregulated after TCF4 knockdown in NPCs in 2D culture. N = 3 replicates (circles), with parent #4 cells. d SOX3 relative expression in post-mortem PTHS cortex sample (PTHS #6). N = 3 replicates per group. e Left: Immunostaining for SOX3 in post-mortem PTHS cortex sample (higher magnification images on the right, showing colocalization with DAPI). Right: Quantification of SOX3+ cells in post-mortem PTHS sample. N = 4 sections per group. f Treatment of PTHS NPCs in 2D culture with CHIR99021 rescues SOX3 expression. N = 4 pairs (symbols). g SOX3 knockdown reduces NPC proliferation in 2D culture. N = 3 replicates (circles), with pair #4 cells. h Immunostaining for SOX2 and MAP2 in neurons in 2D culture (2 months in neuronal medium). i Ratio between neurons (MAP2+) and NPCs (SOX2+) in neuronal 2D cultures. N = 4 pairs (symbols). j SOX4 expression is reduced in IP-Glut and N-Glut cells of PTHS CtOs at 8 weeks in vitro. N = 717 (parent) and 382 (PTHS) IP-Glut cells, or 1401 (parent) and 380 (PTHS) N-Glut neurons from pair #4. k SOX4 expression is reduced in PTHS NPCs in 2D culture. Top: TPM expression. Bottom: Relative expression (RT-qPCR). N = 4 pairs (symbols). l Immunostaining for SOX2 and MAP2 in differentiating neuronal 2D cultures after SOX4 knockdown (2 months in neuronal medium). m Ratio between MAP2+ and SOX2+ after SOX4 knockdown in differentiating neuronal 2D cultures. N = 6 (parent) or 7 (PTHS) replicates, with cells from pairs #1 and #4 (symbols). n Ratio between MAP2 and SOX2 gene-expression levels after SOX4 knockdown in differentiating neuronal 2D cultures. N = 4 replicates, with cells from pair #4. Symbols in bar graphs indicate parent-patient identities: diamonds, pair #1; squares, pair #2; triangles, pair #3; circles, pair #4; gray dots, post-mortem samples. Colors in bar graphs, dot or violin plots represent parents (orange), genetically manipulated parents (yellow), PTHS (blue), pharmacologically treated PTHS (light blue), or control post-mortem sample (black) groups. Bar graphs represent mean + SEM. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA (f, g), Kruskal–Wallis H test (j), or two-sample Welch’s t test in remaining panels. Scale bars are 100 µm. DAPI nuclear staining in blue. See Supplementary Data 1 for sample and effect sizes and exact p-values.
Fig 2: Model of dysregulated pathways underlying PTHS pathophysiology.Mechanistic model to explain aberrant cellular phenotypes in PTHS neural structures. Due to TCF4 loss-of-function in PTHS, Wnt signaling activity diminishes, in turn leading to decreased SOX3 expression in NPCs, impairing proliferation. Moreover, we observed that SOX4 is also downregulated in PTHS cells, which we suggest impairs neuronal differentiation and content in the PTHS neural tissue.
Fig 3: SOX3 was a direct target of miR-491-5p. (A) Binding sites between SOX3 and miR-491-5p predicted by bioinformatics analysis. (B) Luciferase activity of SOX3-WT in OC cells considerably reduced by miR-491-5p mimics as indicated by luciferase reporter assay. (C) Relative SOX3 expression in OC patients. (D) Relative SOX3 expression in cancer cell lines. (E,F) Real-time PCR analysis and Western Blot assay for determining SOX3 expression in cells with rescue assay. All tests were conducted for at least three times. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-SOX3 antibody