Strain-induced nuclear translocation of active β-catenin requires GSK-3β inhibition and Akt-dependent pathway.

<p>(A) Cells were pre-incubated with LiCl (20 mM) for 1 h and then subjected to1 h tensile strain. Western blots results showed levels of total and phospho-GSK-3β, nuclear total and active β-catenin, Runx2 and Osx. Runx2 and Osx were normalized to β-actin. Phospho-GSK-3β and active β-catenin were normalized to their respective total protein. The graph represented relative intensities of the active β-catenin bands normalized to total β-catenin, as measured by densitometry. (B) Cells were pre-incubated with Akti-1/2 (40 μM) for 1 h and then subjected to1 h tensile strain. Western blots results showed levels of the phosphorylation of Akt and GSK-3β. The graph represented relative intensities of phospho-GSK-3β bands normalized to total GSK-3β, as measured by densitometry. (C) Cells were pre-incubated with Akti-1/2 for 1 h and then subjected to tensile strain for 1 h before being fixed. Cellular distribution of active β-catenin (green staining) and nuclei counterstained with DAPI (blue staining) were determined by immunofluorescence and confocal microscopy. Scale bar, 50 μm. Each experiment was performed three times individually. Results are presented as the mean ± SEM, * indicates significantly different than controls (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Effects of calcium ionophore or LiCl on osteoblastic mechanoresponses in PC1 deficient osteoblasts under mechanical strain conditions.

<p>(A) PKD1-shRNA cells were pre-incubated with A23187 (200 μM) for 1 h and then subjected to 1 h tensile strain. The phosphorylation of Akt, GSK-3β and the expression of nuclear active β-catenin were measured by western blots. (B) PKD1-shRNA cells were pre-incubated with A23187 (200 μM) or LiCl (20 mM) for 1 h and then subjected to 2 h tensile strain followed by EdU incorporation (20 μM) for 1 h. Analysis for proliferation rate of cells was carried out. (C) PKD1-shRNA cells were pre-incubated with A23187 (200 μM) or LiCl (20 mM) for 1 h. Cytochemical staining for alkaline phosphatase (ALP) was performed after 1 h tensile strain. Each experiment was performed three times individually. Results are presented as the mean ± SEM, * indicates significantly different than controls (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Relative expression levels of PKD1 in different groups as detected by Real-time PCR.

<p>There were no significant differences between the non-infected and con-shRNA group with respect to the level of PKD1 mRNA. Compared with Con-shRNA group, the mRNA levels of PKD1 in lentivirus-shRNA#1, 2, 3 and 4 subgroups were reduced by 30.9%, 69.1%, 42.6%, and 47.9%, respectively. Each experiment was performed three times individually. Results are presented as the mean ± SEM, * indicates significantly different than controls (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Association between GSTM1 and GSTT1 Allelic Variants and Head and Neck Squamous Cell Cancinoma

<div><h3>Backgrounds</h3><p><em>GSTM1</em> and <em>GSTT1</em> are involved in the detoxification of carcinogens such as smoking by-products, and polymorphisms in these two genes with a result of loss of enzyme activity may increase risk of carcinogenesis. Although many epidemiological studies have investigated the association between <em>GSTM1</em> or <em>GSTT1</em> null genotype and head and neck squamous cell carcinoma (HNSCC), the results remain conflicting. To elucidate the overall association of <em>GSTM1</em>, <em>GSTT1</em> and HNSCC, we included all available studies and performed this meta-analysis.</p> <h3>Methodology/Principal Findings</h3><p>A dataset including 42 articles for <em>GSTM1</em>, 32 articles for <em>GSTT1</em>, and 15 articles for <em>GSTM1</em> and <em>GSTT1</em> in combination were identified by a search in PubMed. Associations beween HNSCC and polymorphisms of <em>GSTM1</em> and <em>GSTT1</em> alone and in combination were analysed by software RevMan 5.1. Stratification analysis on ethnicity and smoking status, sensitivity analysis, heterogeneity among studies and their publication bias were also tested. Association was found in overall analysis between HNSCC and <em>GSTM1</em> and <em>GSTT1</em> null genotype. Stratified by ethnicity, we found increased risks of HNSCC in carriers with <em>GSTM1</em> null genotype in Asian, <em>GSTT1</em> null genotype in South American, and dual null genotype in European and Asian. When stratified by smoking, a more significant association of <em>GSTM1</em> null genotype with HNSCC risk was observed in smokers.</p> <h3>Conclusions/Significance</h3><p>This meta-analysis presented additional evidence of the association between <em>GSTM1</em> and <em>GSTT1</em> polymorphisms and HNSCC risk.</p> </div

Genotype distribution of <i>GSTM1</i> and <i>GSTT1</i> in different Ethnicities.

<p>Abbreviations: ^a, number of carriers with null genotype/ total number; ^b, odds ratio; ^c, confidence interval; ^d, value for heterogeneity; OR, odds ratio; 95% CI, 95% confidence interval.</p>**<p>P <0.01; ^* 0.01≤P<0.05</p

Sense and anti-sense primers for Real-time PCR.

<p>Sense and anti-sense primers for Real-time PCR.</p

Genotype distribution of <i>GSTM1</i> and <i>GSTT1</i> in different smoking status.

<p>Abbreviations: ^a, number of carriers with null genotype/total number; ^b, odds ratio; ^c, confidence interval; ^d, value for heterogeneity; OR, odds ratio; 95% CI, 95% confidence interval</p>*<p>0.01≤P<0.05</p

Forest plot of <i>GSTM1</i> and <i>GSTT1</i> associated with HNSCC under random-effects models.

<p>The diamond shows the overall risk and the line represent the 95% CI for each meta-analysis. Events: null genotype.</p

Effects of mechanical strain on osteoblast differentiation and PKD1 expression.

<p>(A) MC3T3-E1 cells were subjected to tensile strain for different time points (0–4 h), and the cellular protein were immunoblotted for Runx2, Osx, and OCN. Beta-actin was used as loading control. Total RNA was subjected to real-time quantitative PCR analysis for the gene expression of (B) Runx2, (C) Osx, (D) OCN and (E) PKD1. Messenger RNA expression is calculated as a ratio to the GAPDH mRNA level and expressed relative to non-strained control cells. Each experiment was performed three times individually. Results are presented as the mean ± SEM, * indicates significantly different than controls (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Strain-induced osteoblast differentiation needs PC1.

<p>(A) Effect of 1 h tensile strain on mRNA expression of PKD1, Runx2, Osx, OCN and OPN in con-shRNA and PKD1-shRNA cells. Messenger RNA expression was calculated as a ratio to the GAPDH mRNA level. (B) ALP concentrations in con-shRNA and PKD1-shRNA cells were examined after 1 h tensile strain (C) The protein levels of PC1, Runx2, Osx, OCN and OPN in con-shRNA and PKD1-shRNA cells after subjected to 1 h tensile strain. Beta-actin was used as loading control. (D) Runx2 levels were examined by immunofluorescence and confocal microscopy after 1 h tensile strain. Merged images of Runx2 (green staining) and nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue staining) were shown in the bottom. Scale bar, 50 μm. The experiment was repeated in triplicate. Results are presented as the mean ± SEM, * indicates significantly different than controls (*, p<0.05; **, p<0.01; ***, p<0.001).</p