NSCLC cells do not respond to exogenous Shh but can secrete Shh ligand.

<p>NSCLC cells were treated or not with recombinant Shh (500 ng/ml) at the indicated times. RT-qPCR was performed to evaluate Gli1 and Ptch1 mRNA levels upon treatment in A549 cells (<b>A</b>) and H520 cells (<b>C</b>). Results are presented as fold differences of mRNA levels (2^∧∧Ct) compared with non-treated cells for each time point. Western blot was performed to evaluate Gli1 and Ptch1 protein levels in A549 cells (<b>B</b>) and in H520 cells (<b>D</b>) treated or not with Shh. ß-actin was used as a loading control. Secretion of human Shh was evaluated in the supernatants of A549 and H520 cells by ELISA (<b>E</b>) and confirmed by western blot using an antibody recognizing the secreted active form of Shh (<b>inset E</b>). The western blot was performed with H520 supernatant (H520 sup.) and with recombinant Shh (Rec. Shh) used as a positive control. <b>(F</b>) The knockdown of Shh gene by siRNA was realized in H520 cells. Shh secretion was evaluated by ELISA and expressed in percentage as relative secretion compared with cells transfected with a negative control siRNA (NC) having no homology in vertebrate transcriptome. **p<0,05 (G) After silencing of Shh, NSCLC cells were treated or not with recombinant Shh (500 ng/ml). RT-qPCR was performed to evaluate Gli and Ptch1 mRNA levels. Results are presented as fold of mRNA differences (2^∧∧Ct) in treated cells compared with non-treated cells.</p

Choline-containing metabolite spectrum.

<p>Spectral patterns for the metabolites CHO, PHC and GPC simulated using VeSPA (<a href="https://scion.duhs.duke.edu/vespa/project" target="_blank">https://scion.duhs.duke.edu/vespa/project</a>). Metabolites were simulated at a field strength of 3T (main field 123.25MHz) using a PRESS pulse sequence (TE1 = 20ms, TE2 = 10ms).</p

Shh pathway affects lung fibroblast migration, invasion and collagen synthesis.

<p>(<b>A</b>) Accumulated distance of migration of primary human lung fibroblasts treated with Shh (500 ng/ml) or cyclopamine (10 µM) and monitored by live cell microscopy for 48 hours. The accumulated distance of migration of each cell was determined using ImageJ. ***p<0,01. (<b>B</b>) Scratch wound assay was performed in lung fibroblasts CCL206 treated or not with Shh at the doses indicated or with 10 µM cyclopamine (cyclop) for up to 48 hours. Migration of the cells was recorded using live cell microscopy and representative pictures at 1,5, 12,5 and 26 hours are shown. The colored lines indicate the border of cell migration in each case. (<b>C</b>) The area of the wound was quantified after 26 hours and the percentage of wound closure, relative to the initial area of scratch for each case, was determined. (<b>D</b>) Transmembrane invasion assay was performed in lung fibroblasts treated with Shh (500 ng/ml) or with cyclopamine (10 µM). (<b>E</b>) RT-qPCR was performed to assess MMP2 and MMP9 expression in fibroblasts treated or not with Shh (500 ng/ml) for 48hr. Results are presented as fold of mRNA levels in treated cells compared with non-treated cells. *p<0,1. (<b>F</b>) The total collagen content of lung fibroblasts treated with Shh (500 ng/ml) or TGF-ß1 (5 ng/ml) for the indicated times was quantified using the Sircol collagen assay. *p<0,1, **p<0,05.</p

Choline-containing metabolite amplitude changes in synthetic data.

<p>Amplitudes of choline (CHO, red) and the combined phosphocholine/glycerophosphocholine peak (PHC/GPC, blue) estimated using the MOD_CHOsep (top) and MOD_CHOglobal (bottom) models in synthetic data. Estimated levels of CHO (red) and PHC/GPC (blue) show an increase in line with the simulated levels of CHO and PHC. Also shown are the levels of NAA and CRE and GPC (right panel). Note that the bottom panel shows combined amplitudes for choline-containing compounds as the MOD_CHOglobal model does not differentiate between CHO and other choline-containing metabolites.</p

Shh mediates NSCLC/lung fibroblast reciprocal crosstalk.

<p>CCL206 lung fibroblasts were cultured or not with the supernatant of H520 transfected with a NC siRNA(Sup) or with Shh siRNA (Sup siShh) for 48 h. (A) Levels of secreted Leukemia Inhibitory Factor (LIF) were evaluated in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. *p<0,1; **p<0,05 (B) Levels of secreted Vascular Endothelial Growth Factor (VEGF were assessed in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. **p<0,05 (C) A549 and H520 cells were pre-stained with Hoechst (1 µg/ml) and then cultured for 48 h with CCL206 lung fibroblasts pre-treated with Shh (500 ng/ml) or with SAG (3 nM). The number of Hoechst positive cells was evaluated by fluorescent microscopy and is presented in percentage as relative number of cells compared with the number of cancer cells cultured alone. (D) NSCLC cells A549 and H520 were pre-stained with Hoechst (1 µg/ml) and then co-cultured for 72 h either alone or with CCL206 pre-treated with Shh (500 ng/ml) or with 3 nM SAG. The number of NSCLC cells transmigrating is expressed in percentage as the relative number of cells migrating compared with the total number of cancer cells per transwell. *p<0,1; ***p<0,01.</p

Inhibition of Hedgehog signaling reduces the proliferation of NSCLC cells.

<p>Lung adenocarcinoma A549 cells (<b>A</b>) and lung squamous carcinoma H520 cells (<b>B)</b> were cultured in presence or absence of 10 µM cyclopamine for 5 days. Proliferation was assessed by cell counting and cell survival by MTT assay. *p<0,1; **p<0,05. (<b>C</b>) The Hedgehog-responsive transcription factors Gli1, Gli2 or Gli3 were knocked down with siRNA in A549 cells. RT-qPCR was performed to confirm the specific silencing of each Gli and to assess the expression of the Hedgehog receptor Ptch1. Results are presented as fold differences of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. **p<0,05, ***p<0,01. The impact of silencing Gli1, Gli2 or Gli3 in A549 cell proliferation was assessed by cell counting (<b>D</b>) and in cell survival by MTT assay (<b>E</b>)<b>.</b> Results are presented in percentage as relative proliferation and relative survival compared with cells transfected with the negative control siRNA (NC). *p<0,1<b>.</b> (<b>F</b>) Representative phase-contrast microscopic pictures after 72 hours of siRNA are presented<b>.</b> (<b>G</b>) RT-qPCR was performed to evaluate the effect of the siRNA of Gli1, Gli2 and Gli3 in the expression of the G1/S phase cyclins D (Cyc D1, Cyc D2, Cyc D3) and cyclin E (Cyc E1)<b>.</b> Results are presented as fold of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. *p<0,1; **p<0,05. (<b>H</b>) Western blot of cyclin D2 in A549 cells transfected with Gli siRNA or with a negative control siRNA (NC). Blotting of ß-actin was used as loading control.</p

The choline-acetylcholine cycle.

<p>The enzyme choline acetyltransferase builds acetylcholine (ACH) from choline (CHO) and acetyl-CoA. After ACH is released in the synaptic cleft, the enzyme acetylcholinesterase converts ACH into the inactive metabolites CHO and acetate. After re-uptake into the pre-synaptic terminal, free CHO is phosphorylated into phosphocholine (PHC), a reaction catalysed by choline kinase. PHC is available to mobilise CHO for further ACH production via phospholipase C. CHO is also bound in the cell membrane as phosphatidylcholine (PYC). PHC can be then converted to CDP-choline by CTP:phosphocholinecytidyltransferase. The enzyme CDP-choline:1,2-diacylglycerol cholinephosphotransferase then converts the CDP-choline into phosphatidylcholine (PYC) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171338#pone.0171338.ref020" target="_blank">20</a>]. PYC can also be broken down (via phospholipase C) into glycerophosphocholine (GPC), phosphocholine (PHC), and finally CHO (and side products), or (via phospholipase D) directly into free CHO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171338#pone.0171338.ref019" target="_blank">19</a>]. In the chemical spectra acquired with magnetic resonance spectroscopy (MRS) CHO, GPC and PHC are the only “visible” metabolites of the CHO cycle.</p

Overview of trial and MRS acquisition timing.

<p>Trial timing (top): Each trial had a total length of 5s and begun with a short onset jitter of 0–0.29 s, followed by a cue (0.5s) and a jittered inter stimulus interval (ISI) of 0.8 to 1.3s. The stimuli were presented for 0.5s followed by a maximum response interval of 2.2s and an inter trial interval (ITI) of 0.2 to 0.5s. MRS acquisition (bottom): We collected two MRS acquisitions for each trial (2.5s)—an early epoch, covering the cue, the attention shift phase and part of the stimuli, and a late epoch covering the rest of the trial.</p

Sum of MRS voxels over all subjects.

<p>For better visualization the MRS voxel masks were transformed to MNI space. All subjects contributed to coordinates with the highest overlap, including the average center coordinate (MNI -18, -72, 42) (yellow color).</p

Reference metabolites and functional control.

<p>There were no effects of condition (ipsilateral, neutral or contralateral) or epoch (attention or baseline), and no condition by epoch interaction on reference and control metabolites: CRE (left), NAA (middle) and GLU (right).</p