Effect of re-expression of E-cadherin on cell motility, invasion and <i>in vivo</i> growth.

<p>(A) Relative motility as determined by the ability of 2008-EV, 2008-Ecad, CLDN3KD, CLDN3KD-Ecad, CLDN4KD and CLDN4KD-Ecad cells to close a wound made by creating a scratch through a lawn of confluent cells. Cell images were taken 750 minutes after the scratch. (B) Relative invasion of cells through a layer of Matrigel coated on the filter of a Boyden chamber measured at 24 h after seeding. Bars represent mean ± SEM from 3 independent experiments. (C) Relative growth rate of 2008-EV (∇), 2008-Ecad (▴), CLDN4KD (◊) and CLDN4KD-Ecad (▪) tumors after SC inoculation of 1×10⁶ cells in nu/nu mice (n = 8 for each tumor type). Vertical bars, ± SEM.</p

Knockdown of CLDN3 or CLDN4 actives the PI3K/Akt pathway.

<p>(A) Knockdown of CLDN3 and CLDN4 in 2008 cells increased Akt phosphorylation. The extent of Akt phosphorylation was measured by Western blot analysis using anti-phospho-Akt antibody and expressed as the ratio relative to that in the scrambled control after normalization to total Akt levels. Values are mean ± SEM, n = 6. **p<0.01 <i>versus</i> scrambled-shRNAi control. (B) Ectopic expression of CLDN3 in HEY cells inhibited Akt phosphorylation. Levels of phosphorylated Akt in the HEY-CLDN3 and HEY-CLDN4 cells were expressed as the ratio relative to that in empty vector counterpart after normalization for total Akt. Values are mean ± SEM, n = 3. **p<0.01 <i>versus</i> empty vector control. (C) Re-expression of E-cadherin in 2008-scb, CLDN3KD and CLDN4KD cells reduced Akt phosphorylation. Levels of phosphorylated Akt in E-cadherin stably transfected 2008-scb, CLDN3KD and CLDN4KD cells were expressed as the ratio relative to that in 2008-scb-Ecad cells after normalization for total Akt. Values are mean ± SEM, n = 3. *p<0.05 and **p<0.01 <i>versus</i> 2008-scb-Ecad control.</p

Effect of rescuing expression of CLDN3 and CLDN4, or re-expression of E-cadherin, on PIP3 level and PI3K activity.

<p>(A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 <i>versus</i> 2008-scb control; ^#p<0.05 <i>versus</i> CLDN3KD; ^##p<0.01 <i>versus</i> CLDN4KD. (B) Activity of PI3K immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 <i>versus</i> 2008-scb control;^#p<0.01 <i>versus</i> CLDN3KD; ^##p<0.01 <i>versus</i> CLDN4KD. (C) PIP3 levels; *p<0.001 <i>versus</i> CLDN3KD; **p<0.001 <i>versus</i> CLDN4KD. (D) Activity of PI3K; *p<0.001 <i>versus</i> CLDN3KD; **p<0.001 <i>versus</i> CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.</p

Forced expression of CLDN3 and CLDN4 in HEY cells decreases N-cadherin and Twist expression.

<p>Representative Western blot analysis showing reduced expression of N-cadherin (A) and Twist (B) in the HEY cells infected with CLDN3- or CLDN4-expressing lentiviruses. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in empty vector control after normalization to α-tubulin or β-actin. Values are mean ± SEM. *p<0.05 <i>versus</i> empty vector control; **p<0.01 <i>versus</i> empty vector control.</p

Effect of re-expression of E-cadherin on expression of N-cadherin and Twist.

<p>Representative Western blot analysis showing reduced expression of N-cadherin (A) and Twist (B) in the CLDN3KD and CLDN4KD cells stably transfected with pcDNA3.1-E-cadherin-GFP vector. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in empty vector control after normalization to β-actin. Values are mean ± SEM, n = 4. **p<0.01 <i>versus</i> empty vector control.</p

CLDN3 and CLDN4 regulate the expression of EMT markers.

<p>(A) Western blot analysis of the expression of E-cadherin and mesenchymal markers N-cadherin and vimentin in the scrambled control 2008 (2008-scb), CLDN3KD and CLDN4KD cells. (B) Western blot analysis of the levels of EMT-inducing transcription factors. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the 2008-scb cells after normalization to α-tubulin. Values are mean ± SEM. *p<0.05 <i>versus</i> 2008-scb; **p<0.01 <i>versus</i> 2008-scb.</p

Relative Risk of worse Disease-Free-Survival in relation to VDR gene polymorphisms (Multivariate Analysis).

*<p>RR: Relative Risk adjusted for tumor characteristics and ethnicity.</p

FADD^−/− ameliorates post-I/R myocardial infarct size.

<p>(A) Representative photograph of TTC stained heart tissue section obtained 24 hours I/R in FADD^−/− and NLC groups. (B) Graphic summery of infarct size expressed as percentage of area-at-risk (AAR) and the size of AAR. n = 8–10 mice/group. **<i>P</i><0.01, FADD^−/− vs. NLC control.</p

Distribution of VDR-FokI, VDR-BsmI, VDR-TaqI and VDR-ApaI genotypes in African-American and Hispanic/Latina Cases and Controls.

<p>Note: For analysis of alleles F and f, B and b, T and t, A and a, each subject was used twice. <b>F</b>- wild type FokI, <b>f</b>- polymorphic FokI; <b>B</b>- wild type BsmI, <b>b</b>-polymorphic BsmI; <b>T</b>- wild type TaqI, <b>t</b>- polymorphic TaqI; <b>A</b>- wild type ApaI, <b>a</b>- polymorphic ApaI.</p>*<p>P-value is significant if P<0.05;</p>†<p>Analysis adjusted for age, BMI and ethnicity;</p>††<p>Analysis adjusted for age and BMI.</p

Summary of Studies on Breast Cancer and VDR polymorphisms.

<p>AA: African-American.</p>*<p>for postmenopausal women.</p>∧<p>for early-onset breast cancer (age<50).</p>#<p>OR adjusted for age at diagnosis, number of live births, age at menarche and menopause.</p>a<p>associated with low risk of breast cancer.</p