Modulation of EGFR activity during the first hours of myoblast differentiation.

<p>All results were normalized to the result obtained for GM conditions. <b>A</b>. Myoblasts were lysed in proliferation (GM) and at different times in differentiation conditions (DM). Western blot analysis of EGFR on whole cells extracts. EGFR expression decreases by 64% after 24 h in DM (n = 5). <b>B</b>. Myoblasts were fixed in proliferation (GM) and at different times in differentiation conditions (DM). Flow cytometry was performed on non-permeabilized myoblasts. For the control condition myoblasts were only incubated with the secondary antibody. A significant decrease of EGFR at the plasma membrane is observed after 9 h in DM (n = 5). To control the specificity of the antibody against EGFR, we verified that the antibody did not bind any more to myoblasts transfected with a siEGFR. <b>C-D-E</b>. Total EGFR, phospho-p42/p44 MAPK and Myogenin expressions were assessed by Western blot. α-Tubulin expression was used as a loading control. <b>C.</b> Efficiency of EGFR inhibitors. Myoblasts were cultured in GM for 1 h with AG1478 at 10 µM or PD153035 at 3 µM. Western blot analysis shows a significant decrease of phospho-p42/p44 MAPK expression but no difference of EGFR expression (all n = 3). <b>D</b>. Myogenin expression after 24 h treatment with EGFR inhibitors in proliferating conditions (n = 4). <b>E</b>. Phospho-p42/p44 MAPK and EGFR expression during the first hour of differentiation (n = 5).</p

Kir2.1 is activated by EGFR knockdown in myoblasts.

<p>Myoblasts were transfected with either control siRNA or siEGFR. <b>A</b>. Percentages of transfected myoblasts with functional Kir2.1 channels, 48 hours post-transfection. <b>B.</b> Current densities of the total population of myoblasts (including myoblasts with no current, i.e. <5 pA). <b>C.</b> Current-voltage relationships of a myoblast transfected with siEGFR. Voltage-steps were to −40, −60, −80, −100, −120 and −140 mV from a holding potential at −60 mV. The inset shows a control myoblast with no Kir2.1 current, a typical Kir2.1 current recorded from a myoblast, 48 h after transfection with siEGFR. Addition of 500 µM Ba²⁺ inhibited this current. <b>D.</b> Myoblasts were first transfected with control siRNA or siEGFR, and 24 h later with a plasmid coding for GFP-Kir2.1. One day after, immunoprecipitation of GFP was performed. Immunoblots reveal Kir2.1 and phospho-tyrosine (P-Tyr). <b>E</b>. Cytoplasmic Ca²⁺ was assessed with Fura-2-AM on proliferating myoblasts, 2 days after siRNA transfection. Intracellular Ca²⁺ stores were depleted with 10 µM thapsigargin (Tg) in a medium containing 250 nM Ca²⁺. Then 1.8 mM Ca²⁺ was subsequently added to reveal SOCE. The first part of the experiment was performed with a medium containing 30 mM KCl in order to clamp cells at around −40 mV. The second part was performed with a medium containing 5 mM KCl allowing cells to hyperpolarize. <b>F</b>. Quantification of peak SOCE (n = 6; * p<0.05).</p

Vitamin K3 prevents differentiation without inducing proliferation.

<p>A. Myoblasts were cultured either in GM or in DM ±10 µM vitamin K3. Differentiation was assessed by immunostaining (MEF2 in red; MyHC in green; nuclei in blue with DAPI). B. Quantification of the immuno-fluorescence shown in A (n = 4). C. Vitamin K3 in differentiation conditions for 24 h does not increase cell proliferation as assessed by the average of the number of nuclei (DAPI) per field (from 3 independent experiments; p = 0.4).</p

Myoblast differentiation is induced by EGFR knockdown.

<p>Proliferating myoblasts were transfected either with a control siRNA or with a siEGFR, and then maintained in proliferation condition for 5 days. <b>A.</b> Total cell lysates were analyzed by Western Blot. Myogenin and MEF2 expressions were used as early markers of differentiation, Myosin heavy chain (MyHC) as a late marker of differentiation, and α-Tubulin as a loading control. <b>B</b>. Differentiation of myoblasts was observed by the staining of MEF2 (red) and MyHC (green), and nuclei by DAPI (blue). Cesium (Cs 10 mM) was added to the medium to block the hyperpolarization. Myotubes resulting from myoblast fusion were observed (white arrows). <b>C.</b> Quantification of the immuno-fluorescence shown in B (n>3).</p

EGFR expression is down-regulated during myoblast differentiation.

<p>Cells were cultured in either growth medium (GM) or differentiation medium (DM) for 24 h. <b>A</b>. Phospho-RTK arrays were hybridized with 500 µg proteins of whole cell lysates and detected with an anti-phosphotyrosine antibody. Quantification of each spot was performed and expressed in arbitrary units (AU). The results for the ErbB receptors are shown (n = 2). <b>B</b>. EGFR and CD56 expression using FACS technique. Myoblasts were fixed and non-permeabilized (n = 5). <b>C.</b> EGFR expression was confirmed by Western Blot (whole cell lysates). Myogenin and MEF2 expressions were used as differentiation markers, and α-Tubulin as loading control. <b>D</b>. Efficiency of the siRNA against EGFR. Myoblasts were transfected either with a control siRNA or with a siEGFR, and then maintained in proliferation condition for 24 h. EGFR expression was determined by Western Blot (a representative result is shown, n>5).</p

GIRK channels are present on hcrt/orx neurons.

<p>(A) Subtraction of the current-voltage relationships obtained at voltages from −150 to +10 mV before (ACSF) and during application of baclofen at 10 µM. revealed a baclofen-dependent inward current that reversed around −55 mV, which is the inversion potential of potassium in the experimental conditions. (B) In SDC, subtraction of the current-voltage relationships from −150 to +10 mV measured successively in presence of baclofen alone (baclofen) and following subsequent addition of clonidine at 10 µM demonstrated that baclofen occluded the effect of clonidine.</p

Effects of clonidine on hcrt/orx neurons in control and sleep deprived conditions.

<p>(A) In CC, as evident by subtraction of the current-voltage relationships obtained at voltages from −150 to +10 mV before (ACSF) and during application of clonidine at 10 µM, bath-application of clonidine did not elicit any current in hcrt/orx neurons. (B) In SDC, in contrast, subtraction of the currents measured in presence and absence of clonidine revealed a clonidine-dependent inward potassium current. Indeed, the inversion potential is around −55 mV which is the inversion potential of potassium in the experimental conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016672#s4" target="_blank">Methods</a>).</p

Effects of clonidine on identified hcrt/orx neurons.

<p>(A) Experimental protocol. Animals are maintained in a 12 h light/dark cycle (lights on from 8:00 am to 8:00 pm). After a normal cycle of sleep and waking, rats were either allowed to sleep from 8:00 to 10:00 am or kept fully awake; being gently sleep deprived for the same 2 hours interval. Recordings are made between 11 am and 1 pm in neurons obtained from rats which had slept (control condition, CC) or had been sleep deprived (sleep deprived condition, SDC) between 8 and 10 am. (B) Absence of effect of 10 µM clonidine in CC. (C–D) In SDC hcrt/orx neurons are inhibited by clonidine at both 100 nM (C) and 10 µM (D). (E) Idoxazan at 10 µM impedes the inhibitory and hyperpolarizing effect of clonidine in SDC. (F) Barium at 200 µM impedes the inhibitory and hyperpolarizing effect of clonidine in SDC.</p