Human <i>SOX3</i> promoter is hypomethylated during early phases of neural differentiation of NT2/D1 cells.

<p>(A) CpG islands localization within the human <i>SOX3</i> promoter determined by MethPrimer online software. Arrows I and II indicate CpG island regions. Bellow MethPrimer graph is a schematic representation of the human <i>SOX3</i> promoter and regions analyzed using MSP and pyrosequencing (Pyro S). Individual CpGs are represented as black dots. Numbers represent end points of the analyzed promoter region relative to tss (+1). (B) Analysis of <i>SOX3</i> promoter methylation in untreated (NT2/D1) and cells treated with RA in indicated time points (2, 4 and 7 days) by MSP. Product obtained with primers corresponding to methylated (M) 2^nd CpG island within <i>SOX3</i> promoter and product obtained with primers corresponding to unmethylated (U) 2^nd CpG island within <i>SOX3</i> promoter were separated on agarose gel. (C) Quantitative analysis of <i>SOX3</i> promoter methylation in untreated (NT2/D1) and cells treated with RA in indicated time points (2, 4 and 7 days) by pyrosequencing of the 2^nd CpG island. Bars indicate mean levels of methylation of <i>SOX3</i> promoter in each time point. (D) Effects of 1μM 5-azaC treatment on the expression of SOX3 protein in NT2/D1 cells. Quantity of SOX3 protein in treated cells was calculated relative to untreated NT2/D1 cells (set at 1) and presented as the means ± S.D. of at least three independent experiments; *P<0.05. Caspase-3 expression was used as a positive control. Representative blots are shown.</p

Immunocytochemical co-localization of SOX3 and OCT4 during early phases of RA-induced neural differentiation of NT2/D1 cells.

<p>Immunocytochemical detection of SOX3 and OCT4 in untreated NT2/D1 cells (A1-A4) and NT2/D1 cells treated with RA for 2 (B1-B4), 4 (C1-C4), and 7 (D1-D4) days. Cells with high level of SOX3/low level of OCT4 expression are designated by white arrowheads in panels B1-B4. Cells with low level of SOX3/high level of OCT4 expression are designated by yellow arrowheads in panels B1-B4. Cells that are highly imunopositive for both markers are designated by white arrows in panels C1-C4. Cell nuclei were stained with DAPI (A3, B3, C3, D3). Scale bar: 50 μm.</p

ChIP-qPCR analysis of the <i>SOX1</i> core promoter.

<p>(A) Schematic representation of the human <i>SOX1</i> core promoter analyzed by ChIP. The positions of the region relative to TSS are indicated. (B) ChIP-qPCR results for the indicated histone modifications. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

ChIP-qPCR analysis of <i>SOX3</i> regulatory regions.

<p>(A) Schematic representation of the human <i>SOX3</i> gene indicating <i>SOX3</i> upstream (left tile), <i>SOX3</i> core promoter (center tile) and <i>SOX3</i> downstream (right tile) regions analyzed by ChIP. The positions of analyzed regions, relative to TSS, are indicated. (B-F) ChIP-qPCR results for the indicated histone modification that correspond to <i>SOX3</i> regions presented above. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

Assessment of the role of SOX14 in HeLa cell migration, viability and cell cycle.

<p><b>A</b>- The effect of wt or DN1 SOX14 overexpression on migration of HeLa cells, wound-scratch migration assay. Representative images of cell migration are presented. The graph quantifies migration of the transfected cells 5 h after scratching. The changes in migration distance were quantified by measuring the difference in gap closure where gap width at 0 h was set as 100%. The results are presented as the means ± SEM of at least three independent experiments. <b>B</b>—MTS viability assay performed 24 h after transient transfection of HeLa cells with SOX14wt or SOX14DN1. Relative cell viability was calculated as a percentage of mock transfected HeLa cell viability that was set as 100%. Results are presented as the means ± SEM of at least three independent experiments. P-values were calculated using Student’s t-test, ***p≤0.001. <b>C</b>—Flow cytometry analysis of cell cycle distribution in HeLa cells 24 h after transfection with empty vector (mock), SOX14wt or SOX14DN1. One representative analysis is shown in the left panel, while the results of three independent experiments for cell cycle distribution are presented on dot plot.</p

Immunocytochemical detection of MAP2, GFAP and SOX14 in undifferentiated P19 and differentiated P19-N cells.

<p>Panel I: Immunocytochemical detection of MAP2 and GFAP-positive cells in P19-N. Panel II: Immunocytochemical detection of MAP2 and SOX14-positive cells in P19 and P19-N. The P19-N population consists of a large number of MAP2 terminally differentiated neurons (C and D), and a few GFAP-positive astroglial cells (B and D). Specific SOX14 immunoreactivity/punctated nuclear signal was detected in a majority of cells in differentiated P19-N cultures (I, K and M), and at basal level in P19 cells (F). DIC transmitted light images show morphology of SOX14+ cells in P19-N population (G and H). Yellow arrowhead in G-I marks flat cells with large nuclei which show strong SOX14 immunoreactivity. SOX14 is expressed in MAP2-positive neurons (K, arrows in L and M) and in non-neuronal cells (K, arrowheads in L and M). Boxed regions in J and K are enlarged in the same figures. Cell nuclei were counterstained with DAPI (A, D, E, H, J and L). Scale bars: A–K 50 μm, L and M 20 μm.</p

Generation and analysis of dominant negative forms of SOX14.

<p><b>A</b>- Schematic representation of human SOX14wt protein and two truncated forms of SOX14 protein, lacking the C-terminal domain, SOX14DN2 (142 amino acids in length) and SOX14DN1 (93 amino acids in length). <b>B</b>—PCR analysis of <i>SOX14</i> expression upon transient transfection with empty vector (mock) and vectors expressing SOX14wt or truncated SOX14 protein (SOX14DN1 and SOX14DN2), as indicated. <i>GAPDH</i> was used as the loading control. Three independent experiments were performed, and one representative PCR is shown. M- DNA ladder, C- PCR negative control. <b>C</b>–Functional analysis of dominant negative SOX14 forms, a SOX responsive luciferase reporter plasmid 3xSXluc was transfected into HeLa cells in combination with either empty vector—pcDNA3.1 (mock), pcDNA3.1SOX14 (SOX14), pcDNA3.1SOX14DN1 (DN1) or pcDNA3.1SOX14DN2 (DN2). Normalized luciferase activities were calculated relative to the 3xSXluc activity in cells co-transfected with empty vector, which was set as 100% and relative to the 3xSXluc activity in cells co-transfected with SOX14. Data are presented as the means ± SEM of at least three independent experiments. Mean values of relative luciferase activities were compared with Student’s <i>t</i>-test and P-values calculated *** <i>p</i> ≤ 0.001. <b>D</b>- Competition assay. Increasing amounts of DN1 or DN2 expression vectors (1: 1 and 1: 2) were co-transfected with a fixed amount of SOX14wt expression vector and the luciferase reporter plasmid 3xSXluc. Normalized luciferase activities were calculated as percentages of the 3xSXluc activity in cells co-transfected with SOX14, which was set as 100%. Data are presented as the means ± SEM of at least three independent experiments. Mean values of relative luciferase activities were compared with Student’s <i>t</i>-test and P-values calculated *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001.</p

ChIP-qPCR analysis of the <i>SOX2</i> core promoter.

<p>Schematic representation of the human <i>SOX2</i> core promoter analyzed by ChIP. The positions of the region relative to TSS are indicated. (B) ChIP-qPCR results for the indicated histone modifications. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

The effect of HH pathway inhibition on proliferation, viability, migration and <i>SOX18</i> expression in HeLa cells.

<p><b>a)</b> The inhibitory effect of cyclopamine on GLI1 and PTCH expression. <b>b)</b> Proliferation curve of HeLa cells. Cells were treated with 10μM cyclopamine or tomatidine as a negative control, and counted after 1, 3 and 5 days of continuous treatment. Results were presented as the means ± SEM of at least three independent experiments. <b>c)</b> MTT viability assay performed after 1, 3 and 4 days of treatment with 10 μM cyclopamine or tomatidine. Relative cell viability was calculated as a percentage of HeLa cells viability after tomatidine treatment that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. P values were calculated using Student’s <i>t</i>-test, *p ≤ 0.05, **p ≤ 0.01.<b>d)</b> The effect of cyclopamine on cell’s migration, wound-scratch migration assay. Cells migration was quantified 24 h after scratching in constant presence of cyclopamine or tomatidine,by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. <b>e)</b> Relative <i>SOX18</i> expression after cyclopamine treatment detected by qRT-PCR. Relative <i>SOX18</i> expression was presented as percentage of <i>SOX18</i> expression in cells treated with tomatidine that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates. P values were calculated using Student’s <i>t</i>-test, *p ≤ 0.05, **p ≤ 0.01.<b>f)</b> The effect of cyclopamine on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control. The relative SOX18 protein level in HeLa cells upon treatment with cyclopamine was calculated as a percentage of SOX18 level in cells trated with tomatidine which was set as 100%. Data of three independent experiments are presented at histograms as the means ± SEM. Values of p≤0.01 are marked by **.<b>g)</b> The effect of GANT61 on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control.</p

<i>SOX2</i> is down-regulated during early phases of RA induced neural differentiation of NT2/D1 cells.

<p>(A) Real-time PCR analysis of <i>SOX2</i> gene expression in untreated and RA treated NT2/D1 cells (2, 4 and 7 days of induction). Data were normalized by the amount of <i>GAPDH</i> mRNA and presented relative to the corresponding value for untreated cells, and are means ± S.D., *P<0.05 from triplicate data. (B) Western blot analysis of SOX2 protein in whole cell lysates of untreated and NT2/D1 cells treated with RA for 2, 4 and 7 days. SOX2 protein quantities were expressed relative to untreated NT2/D1 cells (set at 1) and presented as the mean ± S.D. of at least three independent experiments, *P<0.05. α-tubulin was used as loading control. Representative blots are shown.</p