Age-Adjusted Suicide Rates at the Population Level

<div><p>(A) Age-adjusted suicide rates (per 100,000) for the total population from 1960 to 2002 and fluoxetine prescribed numbers (in millions) from 1988 to 2002</p> <p>(B) Age-adjusted suicide rate predictions for the total population. The solid lines trace out the posterior median model predictions and the dashed lines depict the 95% Bayesian credible intervals. The top red line depicts the predicted suicide rates without fluoxetine and the bottom black line represents the actual rates with fluoxetine.</p> <p>(C) This figure demonstrates the linear relationship between suicide rates and fluoxetine prescription numbers.</p></div

Estimated Number of Lives Saved Since 1988–2002 with the Advent of SSRIs

<p>Data are shown as a posterior median prediction with 95% Bayesian credible intervals.</p

Age-Adjusted Suicide Rates by Sex

<div><p>(A and B) Suicide rates (per 100,000) for the total female (A) and male (B) populations from 1960–2002.</p> <p>(C and D) Age-adjusted suicide rate predictions for the female (C) and male (D) populations. Solid lines trace out the posterior median model predictions and the dashed lines depict the 95% Bayesian credible intervals. The top red line depicts the predicted suicide rates without fluoxetine and the bottom black line represents the current rates with fluoxetine.</p></div

Leptin increased stem cell characteristics of breast cancer cells.

<p>Effect of leptin treatment (14 d in low serum conditions) on: aldefluor activity in <b>(a)</b> MCF7 (n = 7) and <b>(b)</b> MCF10AT1 (n = 6) breast cancer cells; CD44+/CD24- cell population in <b>(c)</b> MCF7 (n = 6) and <b>(d)</b> MCF10AT1 (n = 6) cultures; mammosphere formation by <b>(e)</b> MCF7 (n = 6) and <b>(f)</b> MCF10AT1 (n = 8) cells. (c-f) Leptin treatment was 200 ng/ml. (ANOVA was used to compare among groups with Tukey’s multiple comparison test (a and b). Student’s t-test was used to compare two groups (c-f).) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Neutralizing antibody against TGFB1 blocks leptin mediated actions.

<p>Effect of leptin (200 ng/ml), TGFB1 (2.5 ng/ml), Ab-TGFB1 (5 ng/ml), Ab-TGFB1+Leptin or Ab-TGFB1+TGFB1 treatment (all treatments for 14 days) compared with untreated controls on: <b>(a and b)</b> <i>CDH1</i> mRNA expression in <b>(a)</b> MCF7 & <b>(b)</b> MCF10AT1 cells (qPCR was normalised to <i>HPRT1</i> expression and is presented as fold-change compared to untreated control (CTRL)); <b>(c and d)</b> cell migration rate of <b>(c)</b> MCF7 and <b>(d)</b> MCF10AT1 cells; and <b>(e and f)</b> aldefluor activity of <b>(e)</b> MCF7 and <b>(f)</b> MCF10AT1 cells. (ANOVA was used to compare among groups with Tukey’s multiple comparison test. Data was combined from 2–3 experiments representative of at least 3 independent experiments.) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Increased TGFB1 protein expression in response to leptin.

<p><b>(a and b)</b> FACS histograms showing increased intracellular TGFB1 levels in <b>(a)</b> MCF7 and <b>(b)</b> MCF10AT1 cells after chronic 200 ng/ml leptin treatment. (Red line–untreated control, Blue line–Leptin treated 200 ng/ml for 14 days.) <b>(c and d)</b> Dose responses for TGFB1 protein levels after leptin treatment in <b>(c)</b> MCF7 (n = 6) and <b>(d)</b> MCF10AT1 (n = 6) cells. (ANOVA was used to compare among groups with Tukey’s multiple comparison test.) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Leptin decreased cell aggregation, and increased cell migration and invasion.

<p>MCF7 or MCF10AT1 cells in low serum media were treated for 14 days with 200 ng/ml leptin (LEPT) and compared to untreated controls (CTRL). (<b>a</b>) CTRL and (<b>b</b>) LEPT-treated MCF7 cells are morphologically different. <b>(c)</b> Effect on MCF7 cells by hanging drop cell aggregation assay. ***P<0.001 (n = 5 drops, Student’s t-test. Data shown is from one representative experiment of 3 independent experiments.) <b>(d)</b> Effect on invasion of MCF7 cells into matrigel. ***P<0.001 (n = 18–20 wells, Student’s t-test, data combined from 3 independent experiments). <b>(e) and (f)</b> Effect on migration rate of <b>(e)</b> MCF7 and <b>(f)</b> MCF10AT1 cells measured by wound healing scratch assay. ***P<0.001 (n = 17–24 wells, Student’s t-test, data combined from several independent experiments).</p

Breast cancer cells express LEPR and increase proliferation in response to leptin.

<p><b>(a)</b><i>LEPR</i> mRNA (qPCR, relative to <i>HPRT1</i>) and <b>(b)</b> LEPR protein expression (western blot) in breast epithelial cell lines. (Student’s t-test was used to compare differences in mRNA levels between pairs of cell lines—MCF10A vs MCF10AT1 and MCF7 vs MDA-MB-231. n = 9–11 wells combined from several experiments.) ***P<0.001. <b>(c)</b> MCF7 and <b>(d)</b> MCF10AT1 cells increased proliferation in response to 72 hr of acute leptin treatment, as measured by MTT assay. (n = 6–8 wells. ANOVA was used to compare among groups with Tukey’s multiple comparison test. **P<0.01, ***P<0.001 compared to control (CTRL)).</p

Sequences of primers used for quantitative RT-PCR.

<p>Sequences of primers used for quantitative RT-PCR.</p

Leptin and TGFB signalling components are present.

<p><b>(a)</b> mRNA expression of components of LEPR signalling: <i>LEPR</i>, <i>STAT3</i>, <i>C-JUN</i>, and <i>C-FOS</i>; <b>(b)</b> Effect of leptin treatment (200 ng/ml, 14d, low serum) on phosphorylated STAT3 (STAT3-P) levels (with total STAT3 and β-actin loading controls) in MCF7 cells by western blotting. <b>(c)</b> mRNA expression of components of TGFB1 signalling: <i>TGFBR1</i>, <i>TGFBRII</i>, <i>SMAD2</i>, <i>SMAD3</i>; in MCF7 and MCF10AT1 cells (n = 6 per group) (qPCR, relative to <i>HPRT1</i>).</p