Frequencies of suicidal feelings by bullying status in middle(-late) adolescents.

<p><i>Note</i>. Suicidal feelings are significantly associated with bullying status in middle(-late) adolescents (^b<i>p</i><0.001, Kruskal-Wallis test). Severity of suicidal feelings is significantly higher in those bullied (pure victims and bully-victims) than in the uninvolved and pure bullies (^c<i>p</i><0.001, Bonferroni post-hoc test), and the severity in pure bullies is significantly higher than in the uninvolved (^d<i>p</i><0.001, Bonferroni post-hoc test) in middle(-late) adolescents. (The total number of subjects is less than the number of subjects analyzed, due to missing data for suicidal feelings or bullying status.)</p><p>Frequencies of suicidal feelings by bullying status in middle(-late) adolescents.</p

The number of the adolescents who sought help for psychological distress according to bullying status^a and sources of help.

<p><i>Note</i>. The number of students who sought help, divided according to the total number of adolescents, is shown by bullying status. The frequencies of the subjects who sought help are significantly associated with bullying status in the adolescents (^ap<0.001, Kruskal-Wallis test). The frequency is significantly higher among pure victims than among the uninvolved and pure bullies (p<0.001, with Bonferroni test). *Multiple answers.</p><p>The number of the adolescents who sought help for psychological distress according to bullying status^a and sources of help.</p

Interactive effects of suicidal feelings and bullying status on help-seeking for psychological distress in middle(-late) adolescents.

<p><i>Note</i>. Odds ratio for seeking help (adjusted for gender and age); 95% CI = 95% confidence interval; mild  =  having mild suicidal feelings; serious  =  having serious suicidal feelings. Reference  =  having no suicidal feelings. In each section, subjects with missing data were excluded from the statistical analyses. *: <i>p</i><0.05; **: <i>p</i><0.01; ***: <i>p</i><0.001.</p

Effects of suicidal feelings on help-seeking for psychological distress by source of the help, in pure victims.

<p>Note. Odds ratio for seeking help from the source (adjusted for gender and age); 95% CI = 95% confidence interval; mild  =  having mild suicidal feelings; serious  =  having serious suicidal feelings. Reference  =  having no suicidal feelings. *: p<0.05; **: p<0.01.</p

An E2F1-HOXB9 Transcriptional Circuit Is Associated with Breast Cancer Progression

<div><p>Homeobox B9 (HOXB9), a member of the homeobox gene family, is overexpressed in breast cancer and promotes tumor progression and metastasis by stimulating epithelial-to-mesenchymal transition and angiogenesis within the tumor microenvironment. HOXB9 activates the TGFβ-ATM axis, leading to checkpoint activation and DNA repair, which engenders radioresistance in breast cancer cells. Despite detailed reports of the role of HOXB9 in breast cancer, the factors that regulate <i>HOXB9</i> transcription have not been extensively examined. Here we uncover an underlying mechanism that may suggest novel targeting strategies for breast cancer treatment. To identify a transcription factor binding site (TFBS) in the <i>HOXB9</i> promoter region, a dual luciferase reporter assay was conducted. Protein candidates that may directly attach to a TFBS of <i>HOXB9</i> were examined by Q-PCR, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and mutation analysis. A <i>HOXB9</i> promoter region from −404 to −392 was identified as TFBS, and E2F1 was a potential binding candidate in this region. The induction of <i>HOXB9</i> expression by E2F1 was observed by Q-PCR in several breast cancer cell lines overexpressing E2F1. The stimulatory effect of E2F1 on <i>HOXB9</i> transcription and its ability to bind the TFBS were confirmed by luciferase, EMSA and ChIP assay. Immunohistochemical analysis of 139 breast cancer tissue samples revealed a significant correlation between E2F1 and HOXB9 expression (p<0.001). Furthermore, a CDK4/6 inhibitor suppressed E2F1 expression and also reduced expression of <i>HOXB9</i> and its downstream target genes. Our in vitro analysis identified the TFBS of the <i>HOXB9</i> promoter region and suggested that E2F1 is a direct regulator of <i>HOXB9</i> expression; these data support the strong correlation we found between E2F1 and HOXB9 in clinical breast cancer samples. These results suggest that targeting the E2F1/HOXB9 axis may be a novel strategy for the control or prevention of cancer progression and metastasis.</p></div

Identification of transcription factor binding site (TFBS) of <i>HOXB9</i> promoter.

<p>(<b>A</b>) <i>HOXB9</i> promoter region and primer sets used for cloning. (<b>B</b>) Reporter plasmids with the <i>HOXB9</i> promoter region and deletion variants were constructed by inserting the PCR product of the putative HOXB9 promoter region. The numbers of the left side of graph indicate the full length of HOXB9 promoter and deletion variants, the right side shows transcriptional activity of each variant in MDA-MB231 cells. The difference in relative luciferase activity between pGL3-439 and pGL3-358 constructs was recorded. (*p<0.01) (<b>C</b>) Additional deletion plasmids between bps −439 and −358 of the promoter were constructed to identify the exact position of TFBS and gap of gene activity in that region was recorded each time when assay was repeated. The difference in relative luciferase activity between pGL3-404 and pGL3-392 constructs was significant (*p<0.01).</p

Correlation between E2F1 and HOXB9 protein expression in breast cancer tissues.

<p>(<b>A</b>) A cohort of 139 breast cancer clinical samples stained with anti-E2F1 antibody was interrogated for HOXB9 expression. i- E2F1 positive, ii- HOXB9 positive, iii- E2F1 negative, iv- HOXB9 negative staining (original magnification, 100×). (<b>B</b>) The cross-link results of 139 breast cancer clinical samples to evaluate the correlation between E2F1 and HOXB9 staining. (<b>C,D</b>) The data was analyzed by hormone receptor status. (<b>E</b>) Kaplan–Meier plots of clinical outcomes of HR(+)/HER2(−) type breast cancer patients by E2F1 expression. (<b>F</b>) Kaplan–Meier plots of breast cancer patients' clinical outcomes by combination of HOXB9 and E2F1 expression. All p values were calculated using the log rank test.</p

HOXB9/E2F1 staining and breast cancer subtypes.

<p>HR: Hormone receptor.</p><p>HER2: Human Epidermal growth factor Receptor 2.</p><p>Triple neg: Triple negative.</p

Cytokine mRNA Expression by Foxp3⁺CD25⁺ and Foxp3⁻CD25⁺T-cells from long term survivors.

<p>Message levels of TGFβ, IL-10 and IFNγ in CD4⁺Foxp3⁺CD25⁺ T-cells obtained from spleens of long term survivors (DSTx4+LTx rats; survival>100 days) were quantified and compared to those expressed by CD4⁺Foxp3⁻CD25⁺ T-cells obtained from these same animals. Quantitative Real-time (RT)-PCR was used to quantify cytokine mRNA expression in each population. HRPT was used as an endogenous control to normalize each sample. Data represent mean±SD. * P<0.05 compared with Foxp3⁻CD25⁺ T-cells. N = 5 for each group.</p

The correlation of E2F1 and HOXB9 in IHC staining.

<p>The correlation of E2F1 and HOXB9 in IHC staining.</p