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

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

<div><p>The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.</p></div

AR antagonist Bicalutamide reduces AR and Sox2OT levels in mouse E12.0.

<p>AR antagonist Bicalutamide reduces AR and Sox2OT levels in mouse E12.0.</p

AR binds ARSO-Sox2OT chromatin region and allows Sox2OT transcription.

<p><b>(a)</b> AR consensus sites in the chromosome 3 and in Sox2OT ncRNA. Grey arrows indicate DNA region tested by ChIP with ChIP-grade antibodies (ChIP site); vertical red lines indicate AR consensus site (ARE); green arrows indicate the sequence on Sox2OT RNA tested by RIP (RIP site); <b>(b)</b> Representative images showing Chromatin Immuno Precipitation of AR and RNA Polymerase II. The images show PCR of antibody–precipitated E12.0 forebrains and eNSCs chromatins with primers amplifying ARSO-Sox2ot region. The lower panels show the quantification of AR and RNA Pol ChIPs. Values are mean±SEM of ratios between PCR signal intensity of the AR and RNA Pol II antibodies–precipitated sample and input chromatin (IN). Notably, RNA pol II binds ARSO-Sox2ot sequence in an AR-dependent manner. For RNA pol II in E12.0 forebrains: control = 0.51±0.06, Bicalutamide = -0.18±0.06, t-test p = 0.0023. For AR in E12.0 forebrains: control = 0.51±0.13, Bicalutamide = -0.26±0.08, t-test p = 0.0003. For RNA pol II in embryonic NSCs: control = 1.36±0.21, Bicalutamide = 1.28±0.07, t-test p = 0.0002. For AR in embryonic NSCs: control = 1.28±0.07, Bicalutamide = -0.15±0.09, t-test p = 0.000017. Results derived from four independent experiments. Data were analyzed by two-tailed t-test. <b>(c)</b> RNA Immunoprecipitation (RIP) assay of eNSCs and E12.0 forebrains. RNA was subjected to IP assays with anti-AR and anti-Rpb1 CTD phosphorylated at Serine 2 and Serine 5 (RNA Pol II) antibodies or normal rabbit IgG as described in Materials and Methods section. RNA immunoprecipitates and input lysate RNAs were reverse transcription-PCR (RT-PCR)–amplified to measure the abundance of Sox2OT RNA present in the eNSCs and forebrains (control). Agarose gel of RT-PCR products from RIP and input (upper panel). Molecular weight marker sizes (base pair lengths; bps) are shown at the right. Values are mean± SEM of ratios between PCR signal intensity of the AR and RNA Pol II antibodies–precipitated sample and input (lower panel). For RNA pol II in E12.0 forebrains: control = 1.66±0.26, Bicalutamide = -0.60±0.27, t-test p = 0.0084. For AR in E12.0 forebrains: control = 0.51±0.09, Bicalutamide = -0.20±0.16, t-test p = 0,016. For RNA pol II in embryonic NSCs: control = 0.75±0.18, Bicalutamide = 0.16±0.11, t-test p = 0.032. For AR in embryonic NSCs: control = 0.85±0.07, Bicalutamide = 0.13±0.06, t-test p = 0.0002. Results are average of four independent experiments. Data were analyzed by two-tailed t-test. Lane RT+ contains an aliquot of the PCR sample. In the RT- lane, the reverse transcriptase was omitted from the RT reaction. Input are 20% of total RNA. IgG: rabbit IgG as negative control. Bicalutamide treatment were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180579#pone.0180579.g001" target="_blank">Fig 1</a>.</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