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

The effect of SOX14wt and SOX14DN1 on p53 protein in HeLa cells.

<p><b>A</b>- Western blot analysis of p53 protein expression in HeLa cells transfected with either empty vector (mock), SOX14wt or SOX14DN1. GAPDH was used as the loading control. Quantification of the effect of SOX14 overexpression on p53 protein level is presented in the graph. Amounts of p53 protein in transfected cells were calculated as a percentage of the p53 in mock transfected cells, which was set as 100%. Data are given as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test and. P- values calculated, *p ≤ 0.05. <b>B</b>—The effect of SOX14 overexpression on p53 protein level detected by immunocytochemistry. Cells were transfected with empty vector (mock), SOX14wt or SOX14DN1. Cell nuclei were counterstained with DAPI. Scale bar: 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

Assessment of the role of SOX14 in HeLa cell apoptosis.

<p><b>A</b>. Flow cytometry analysis of Annexin-FITC staining and propidium iodide (PI) accumulation in HeLa cells 24 h after transfection with empty vector (mock), SOX14wt or SOX14DN1. Upper panel: One representative analysis is presented in each quadrant. Q1: PI-/Annexin- cells (live cells); Q2: PI-/Annexin V+ cells (early apoptosis); Q3: PI+/Annexin+ cells (late apoptosis); Q4: PI+/Annexin- cells (necrotic cells). Lower panel: Quantitative analyses of PI and Annexin V positive cells are shown on the histograms relative to the control (mock), which was set as 1, as means ± SEM of three independent experiments. Mean values were compared with Student's <i>t</i>-test and P-values calculated, *<i>p</i> ≤ 0.05. <b>B</b>- DAPI staining of HeLa cell nuclei after mock, SOX14wt or SOX14DN1 transient transfection. Cells with fragmented nuclei are indicated by arrows. <b>C</b>- qRT-PCR analysis of <i>Bax</i> and <i>Bcl</i>2 gene expression after transient transfection with empty vector (mock) and vectors expressing SOX14wt or truncated SOX14 protein (SOX14DN1), as indicated. GAPDH was used as the loading control. The effect of SOX14 overexpression on <i>Bax</i> and <i>Bcl</i>2 genes is shown in graphs. The quantities of <i>Bax</i> and <i>Bcl2</i> genes in transfected cells were calculated as percentages of their respective expression levels in mock transfected cells, which were set as 100%. The Bax/Bcl-2 ratio was calculated from their relative mRNA expression. Data are presented as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test and P-values calculated *p ≤ 0.05,**p ≤ 0.01 <b>D</b>—Western blot analysis of cleaved PARP expression in HeLa cells transfected with either empty vector (mock), SOX14wt or DN1 expression construct for 24 h. α- Tubulin was used as the loading control. The effect of SOX14 overexpression on cleaved PARP protein level is presented in the graph. The quantities of cleaved PARP protein in transfected cells were calculated as a percentage of cleaved PARP in mock transfected cells which was set as 100%. Data are shown as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test and P-values calculated, ***p ≤ 0.001.</p

The effect of SOX14 ectopic expression on p21^Waf1/Cip1 expression in HeLa cells.

<p><b>A</b>- Effect of SOX14 ectopic expression on activity of the <i>CDKN1A</i>/p21^Waf1/Cip1 promoter. The plasmid p21-Luc was co-transfected into HeLa cells with pcDNA3.1 vector, SOX14wt, SOX14DN1 or the p53 expression construct. Normalized luciferase activities were calculated relative to the p21-Luc activity in cells co-transfected with pcDNA3.1 vector, which was set as 100%. Data are presented as the mean ± SEM of at least three independent transfections. Mean values of relative luciferase activities were compared with Student's t-test and P-values are ***p ≤ 0.001. <b>B</b>- qRT-PCR analysis of <i>CDKN1A</i>/p21^Waf1/Cip1 gene expression upon transient transfection with empty vector (mock) and vectors expressing SOX14wt or truncated SOX14 protein (SOX14DN1), as indicated. <i>GAPDH</i> was used as the loading control. Quantification of the effect of SOX14 overexpression on <i>CDKN1A</i>/p21^Waf1/Cip1 gene level is presented in the graph. <i>CDKN1A</i>/p21^Waf1/Cip1 gene expression in transfected cells was calculated as a percentage of the expression level in mock transfected cells, which was set as 100%. Data are given as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test. **p ≤ 0.01. <b>C</b>—Western blot analysis of p21^Waf1/Cip1 expression in HeLa cells transfected with either pcDNA3.1 vector (mock) or SOX14wt expression construct, for 24 h, 48 h and 78 h. α- Tubulin was used as the loading control. The effect of SOX14 overexpression on p21^Waf1/Cip1 protein level is presented in graphs. The quantities of p21^Waf1/Cip1 protein in transfected cells were calculated reltive to the amount in cells transfected with pcDNA3.1 vector, which was set as 100%. Data are presented as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test and P-values were calculated, ***p ≤ 0.001. <b>D</b>—Western blot analysis of p21^Waf1/Cip1 expression in HeLa cells transfected with either pcDNA3.1 vector (mock), SOX14wt or SOX14DN1. GAPDH was used as the loading control. The quantitative effect of SOX14 overexpression on p21 ^Waf1/Cip1 protein level is presented in the graph. Amounts of p21 ^Waf1/Cip1 protein in transfected cells were calculated as a percentage of that in cells transfected with pcDNA3.1 vector, which was set as 100%. Data are presented as the means ± SEM of three independent transfection experiments. Mean values were compared with Student's t-test and P-values calculated, *p ≤ 0.05. <b>E</b>—The effect of SOX14 overexpression on p21^Waf1/Cip1 protein level detected by immunocytochemistry. Cells were transfected with empty vector (mock) or SOX14wt. Cell nuclei were counterstained with DAPI. Scale bar: 50 μm.</p

<i>SOX14</i> methylation status and expression analysis in the HeLa cell line.

<p><b>A</b>- Schematic illustration of <i>SOX14</i> promoter and positions of CpG islands 1, 2, 3 and 4 (presented in blue); positions of methylation specific PCR products 1 and 2 (MSP1 and MSP2) are shown in black lines. <b>B</b>- Methylation analysis of the <i>SOX14</i> promoter by methylation specific PCR (MSP) in HeLa cells. <b>C</b>- Methylation analysis of the <i>SOX14</i> promoter by MSP in NT2/D1 cells. M- primer pairs that anneal only to sequences that are methylated before bisulfite treatment; U—primer pairs that anneal only to sequences that are unmethylated. The product corresponding to MSP1 is 183 bp in length, while that corresponding to MSP2 is 118 bp in length. <b>D</b>- RT- PCR analysis of <i>SOX14</i> expression in HeLa and NT2D1 cells. <i>GAPDH</i> was used as a loading control. Three independent experiments were performed and one representative PCR is shown. NT2/D1 cells were used as a positive control for <i>SOX14</i> expression. m- DNA ladder, C- PCR negative control. <b>E</b>–Immunocytochemical analysis of SOX14 expression in HeLa and NT2/D1 cells. NT2/D1 cells were used as the positive control for SOX14 expression. Scale bar: 50 μm. <b>F</b>–Expression of <i>SOX14</i> following treatment with 5-azaC. HeLa cells were treated with 5μM 5-azaC for 72 h. Total RNA was subjected to RT-PCR in order to analyze <i>SOX14</i> mRNA expression. <i>GAPDH</i> was used for normalization. Three independent experiments were performed, and one representative PCR is shown. m- DNA ladder, C- PCR negative control.</p