Expression of neuronal/glial markers and UCP mRNA during neuronal differentiation.

<p>A. Representative Western blots show the time-dependent expression of neuronal (TUJ-1 for young and NF for adult neurons) and astrocyte (GFAP) markers during mESCs differentiation in culture. Gels were loaded with 20 µg protein per lane. B. Real-time PCR analysis of mESCs shows the amount of UCP2, UCP4 and UCP5 mRNA relative to mRNA amounts of the housekeeping gene GAPDH at different time points during neuronal differentiation. Each data point represents the mean value and SD of 3 independent differentiation experiments.</p

Lack of UCP4 expression in Dcx+/NeuN- neuroblasts in the adult subventricular zone (SVZ).

<p>A. Schematic drawing illustrates the localization of the SVZ of the lateral ventricle in adult mouse brain. B–C. Light microscopy analysis of the representative immunohistostained sample shows the distribution of UCP4- and Dcx-positive cells within the SVZ in 50 µm thick coronal sections of adult mouse brain. D. Representative CLSM images of UCP4 (green), Dcx (red) and NeuN (blue) stained with respective antibodies and visualized using Alexa 488, Alexa 594 and Alexa 633 fluorescent dyes.</p

UCP4 expression starts simultaneously with the expression of neuronal markers.

<p>A. UCP2, UCP4 and UCP5 mRNA levels during neuronal development analyzed by quantitative PCR. UCP mRNA levels in mouse head are shown as a ratio to GAPDH at embryonic day 12 (E12; inset) and as a ratio (UCP mRNA)/(GAPDH mRNA) at different days to (UCP mRNA)/(GAPDH mRNA) at E8. B. Representative Western blot indicates the simultaneous start of UCP4 protein expression with the expression of the neuronal marker TUJ-1. C-D. Representative Western blots demonstrate that UCP2 is not present at the protein level in the tested embryonic tissue (C) as well as in young postnatal neocortical brain tissue (NC) (D). Gels were loaded with 20 µg protein per lane. GAPDH, β-actin and VDAC were used as loading controls. At least 3 samples of pooled embryonic and postnatal tissue from at least 6 mice were analyzed at each condition (Experiments A–D).</p

Neuronal differentiation coincides with a suppression of UCP2 and an induction of UCP4 expression.

<p>A–B. Representative Western blots of UCP2 (A) and UCP4 (B) expression during the differentiation of mESCs in culture. Activated T-cells and primary neuronal cultures (13 days) were used as positive controls. Gels were loaded with 20 µg protein per lane. Cells were collected at different time points from at least three independent differentiation experiments. C. Representative fluorescent images showing the time course of UCP4 and MAP2 expression during differentiation of mESCs to neurons. Primary antibodies were visualized by Alexa-488 (MAP2, green) and Alexa-567 (UCP4, red) respectively. Cell nuclei were counterstained with DAPI (blue).</p

Summary of UCP2, UCP4 and UCP5 distribution at mRNA and protein levels, obtained in our laboratory using RT PCR and WB with positive (recombinant proteins) and negative (knockout mouse for UCP2) controls.

<p>Crosses and circles indicate the positive and negative tested tissues of adult mice and murine cells, respectively. X indicates tissues in which UCP2 was always detected. (X) indicates tissues in which UCP2 was often but not always detected or tissues where the protein abundance variation was very strong. Non-analyzed tissues are marked as n.a. Data were published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088474#pone.0088474-Rupprecht2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088474#pone.0088474-Smorodchenko1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088474#pone.0088474-Smorodchenko2" target="_blank">[17]</a> and in the present paper.</p

Embryonic stem cells express the pluripotency marker Oct 3/4 and uncoupling protein 2 simultaneously.

<p>A–E. Representative Western blot images showing UCP expression in mESCs using antibodies against UCP1 (A), UCP2 (B), UCP3 (C), UCP4 (D) and UCP5 (E). Brown adipose tissue (BAT), activated T-cells, brain from adult mice and recombinant mUCP5 were used as positive controls for the respective protein antibodies. Gels were loaded with 20 µg protein per lane. Antibodies directed against VDAC, GAPDH and Hsp 60 were used to visualize the respective proteins as loading controls. mESCs from at least 3 different passages were analyzed in each experiment.</p

The neuroblastoma cell line N18TG2 expresses UCP2 but not UCP4.

<p>A. Representative Western blot analysis of UCP4 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Mouse brain tissue was used as a positive control for the antibody against UCP4. B. Representative Western blot analysis of UCP2 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Thymus of UCP2 knockout (KO) and wild type (wt) mice were used as negative and positive controls for the antibody directed against UCP4. Gels were loaded with 20 µg protein per lane. Cells from at least three different passages were analyzed in each experiment.</p