Addition of exogenous Prl stimulates cell proliferation in a manner which can be blocked by PrlRA.

<p>LAM/TSC cells were cultured in different concentrations of serum (0%, 2%, 10%). Human Prl was added to sub-confluent cells at a concentration of 200 ng/ml, with or without PrlRA (20, 50, 200 ng/ml). After 72 hours, cells were subject to the crystal violet assay. The X-axis depicts serum concentration and the Y-axis shows relative cell proliferation, as determined by absorbance at 600 nm. Each data point represents a triplicate assay. All values are +/- standard deviation, P-value <0.05.</p

Human LAM cells express PrlR, and prolactin stimulates phosphorylation of STAT3 and Erk.

<p>LAM/TSC cells were cultured in serum-free medium overnight. The cells were then exposed to different doses of PrlRA (20, 50, 200 ng/ml) for 15 minutes, and subsequently the cells were exposed to 200 ng/ml Prl for 60 minutes or PBS as a control. Then protein extracts were prepared for Western blot analysis, and probed with antibodies for P-STAT3, STAT3, P-Erk and Erk. (A) Western blot to analyze PrlR in LAM/TSC control cells; an antibody against human PrlR detected a protein band of 89–90 kD. (B) Western blot, using antibodies directed against P-STAT3, STAT3, P-Erk and Erk in LAM/TSC cells following treatment as described in Material and methods section. (C) Densitometric quantification of Western blot signals, in which the Y-axis depicts the ratio between phosphorylated STAT3 and Erk to the total protein.</p

Immunofluorescence of CRL-2620 using PrlR and GHR antibodies.

<p>Cells were transfected with TSC2 or control siRNAs. After over-night incubation, the cells were washed 3 times with PBS and incubated with antibodies for PrlR and GHR. The figures show that PrlR immunostaining was markedly increased following TSC2 knock-down. A strong immunofluorescence signal was observed at intracellular locations. In the negative control panel, cells were incubated with mouse IgG and rabbit IgG antibodies. Fluorescence images were acquired at a magnification of 63x.</p

Detection of PrlR in human LAM cells using immunofluorescence.

<p>Staining LAM/TSC cells using different PrlR antibodies detect a significant immunofluorescence signal in LAM/TSC cells. The antibodies employed were U5 (Thermoscientific) and A12B1 (Invitrogen).</p

Effect of Prl and PrlRA on cell invasion.

<p>LAM/TSC cells were cultured in serum-free medium in 24-well cytoselect transwell plates. The cells were cultured with Prl or Prl and PrlRA (both at 200 ng/ml). After 48 hours, cell extracts were prepared from the layer representing invading cells, and the optical densities of the extracts were measured at 560 nm. At this dose level, Prl did not stimulate cell invasion, but the combination of Prl and PrlRA reduced invasion by 60%; * = P-value <0.05.</p

Lung function, pulmonary manifestations and symptoms of patients according with LAM.

<p>Lung function, pulmonary manifestations and symptoms of patients according with LAM.</p

Age-dependent risk of LAM.

<p><b>(A)</b> on age quartiles in the overall population (<i>p = 0</i>.<i>004</i>) <b>(B)</b> predicted probability of LAM in relationship to age and 95% CI in patients with and without altered pulmonary function tests. Points along the central logistic curve are individual predicted probabilities. Black points refer to patients with normal pulmonary function tests (PFT), white points refer to patients with altered PFT. The corresponding 95% CI for each point appears on the outer logistic curves. The dotted lines refer to 95% CI of predicted probability for patients with altered PFT while the continuous line refers to IC in patients with normal PFT.</p

Demographic and clinical characteristics of enrolled patients according with LAM.

<p>Demographic and clinical characteristics of enrolled patients according with LAM.</p

Population in analysis.

<p>Age is shown as mean ± standard deviation and is referred to first evaluation in the center. LAM = lymphangioleiomyomatosis; TSC = tuberous sclerosis complex; *percentage referred to all adult TSC females patients in which lung scan was available for evaluation.</p

Demographic, pulmonary, clinical characteristic and genetic analysis of TSC patients according with MMPH.

<p>Demographic, pulmonary, clinical characteristic and genetic analysis of TSC patients according with MMPH.</p