Embryonic and neuronal expression of MFSD5 and MFSD11.

<p>MFSD5 antibody labelling in mouse e14-15 embryos are shown in A, with subsequent negative control (B). C depicts the MFSD11 staining in embryo, where D is its negative control. (E) MFSD5 in green and glutamate marker anti-glutaminase in red, co-localized, exemplified in cell indicated by the white arrow. (F) MFSD5 in red and eGFP-marked inhibitory vesicles, VIAAT, in green showed co-localization, depicted by white arrow. (G) MFSD11 in green and glutamate marker anti-glutaminase in red, co-localized, exemplified in cell indicated by the white arrow (H) MFSD11 in red and eGFP-marked inhibitory vesicles, VIAAT, in green showed co-staining, depicted by white arrow. Cell nucleus staining DAPI in blue was included in all staining.</p

Normalized mRNA distribution.

<p>mRNA expression of <i>Mfsd5</i> and <i>Mfsd11</i> in wt mouse brain and peripheral tissues. cDNA from five mice were pooled for each organ. Samples were normalized against the geometric mean between the following reference genes: <i>Gapdh</i>, <i>bTub</i>, <i>Rpl19</i>, <i>Cyclo</i> and <i>Actb</i>. Plots show <i>Mfsd5</i> (A) and <i>Mfsd11</i> (B) expression, with standard deviations (±SD).</p

mRNA expression changes after starvation and HFD of <i>Mfsd5</i> and <i>Mfsd11</i>.

<p>The column charts display the normalized mRNA expression of <i>Mfsd5</i> and <i>Mfsd11</i>, with standard deviations (±SD). Mice were assigned different food; 1) normal chow (control), 2) food-deprived for 24h- and 3) high-fat diet. <i>Mfsd5</i> (A) and <i>Mfsd11</i> (B) expression in cortex, hypothalamus, striatum and brainstem samples from starved and obese mice. cDNA from four mice were pooled per sample. Effects of <i>Mfsd5</i> and <i>Mfsd11</i> expressions in sections from starved mice (n = 4) are depicted in C-D, while HFD (n = 6) samples are shown in E-F. Weight gain of mice in the HFD group compared to controls (I). A schematic description over how the seven brain cuts were made for section analyses (J). The schematic brain was adapted from Allen institute. <i>Gapdh</i>, <i>H3a</i> and <i>Actb</i> were used as housekeeping genes for all analyses. * corresponds to P≤ 0.05, ** to P≤ 0.01 and ***to P≤ 0.001.</p

Abundant MFSD11 protein expression.

<p>DAB-immunohistochemistry stained MFSD11 on 70μm wt floating brain sections. Overview images with specific regions magnified. (A) Staining pattern in cortex, (B) hypothalamic nuclei and the (<b>C</b>) brainstem. The schematic bregma regions were modified from <i>The mouse brain</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156912#pone.0156912.ref028" target="_blank">28</a>].</p

Antibody specificity.

<p>Western Blot was performed on fractionated wt mouse brain and kidney samples to verify antibody specificity. (A) The MFSD5 antibody (Santa Cruz) provided three bands in the brain fraction which correspond to the size of three splice variants of the proteins and the MFSD11 antibody(Sigma-Aldrich) (B) bound to two splice variants in the kidney sample and one in the brain fraction (C). Double immunohistochemistry showed overlap between antibodies from different vendors to verify specificity; MFSD5_SC (Santa Cruz) overlapped with MFSD_MBS (MyBioSource) (D, E) while MFSD11_SA (Sigma-Aldrich) co-stained with MFSD_SC (Santa Cruz) (F, G). The antibodies utilized throughout this manuscript are marked in green. Proximity ligation assay also showed interaction between the two MFSD5 antibodies (H) and the two MFSD11 antibodies (I), while no signal was detected in the negative control (J).</p

Phylogenetic analysis of the human SLCs from the MFS superfamily.

<p>(<b>A</b>) The phylogenetic tree was calculated using RAxML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156912#pone.0156912.ref024" target="_blank">24</a>] with 500 bootstrap replicas. Circles on the nodes indicates the bootstrap support with black as 100% - 95%, grey 94% - 75% and white 74% - 50%. Nodes without circles had less than 50% bootstrap support. Species abbreviations: ac, <i>A</i>. <i>carolinensis</i>; ce, <i>C</i>. <i>elegans</i>; ci, <i>C</i>. <i>intestinalis</i>; cs, <i>C</i>. <i>savignyi</i>; dm, <i>D</i>. <i>melanogaster</i>; dr, <i>D</i>. <i>rerio</i>; gg, <i>G</i>. <i>gallus</i>; ga, <i>G</i>. <i>aculeatus</i>; no prefix, <i>H</i>. <i>sapiens</i>; mm, <i>M</i>. <i>musculus</i>; tn, <i>T</i>. <i>nigviridis</i>. (B) Schematic representation of the branching order obtained in the phylogenetic analysis.</p

<i>Slc38a9</i> gene expression in mouse brain following starvation and high-fat diet.

<p>Relative <i>Slc38a9</i> mRNA expression ±SD are plotted and compared with controls (Unpaired t-tests, significance adjusted for multiple testing, *p≤0.049375, **p≤0.009975, ***p≤0.001, n represents technical replicates). (a) Control group (white bars), Starved group (light grey bars), n = 6 for both groups and all tissue tested, except n = 5 for starved group in cerebellum, Brainstem (p = 0.0442), Cerebellum (p = 0.4865), Cortex (p = 0.0055), Hypothalamus (p = 0.9803). (b) Control group (white bars), High fat diet group (dark grey bars), n = 6 for both groups and all tissue tested, Brainstem (p = 0.9111), Cerebellum (p = 0.6745), Cortex (p = 0.5967), Hypothalamus (p = 0.0014). (c) Control group (white bars, (region I, II, III and VI, n = 2, region IV, V and VII, n = 3), Starved group (light grey bars, (n = 3 for all regions except for regions I and VII were n = 2)). (I (p = 0.0310), II (p = 0.0297), III (p = 0.0009), IV (p = 0.0159), V (p = 0.0743), VI (p = 0.6162) and VII (p = 0.8486)). (d) Control group (white bars, (region I, II, III and VI, n = 2, region IV, V and VII, n = 3)), High-fat diet group (dark grey bars, (n = 3 for all brain regions except for region V, n = 2)). (p = 0.0016), II (p = 0.0007), III (p<0.0001), IV (p = 0.0006), V (p = 0.0266), VI (p = 0.0124) and VII (p = 0.0151). (e) Representation of the mouse brain displaying the brain regions I-VII, with corresponding Bregma numbers (mm).</p

Description of the genomes used for MFSD5 and MFSD11 phylogenetic analysis.

<p>Description of the genomes used for MFSD5 and MFSD11 phylogenetic analysis.</p

SLC38A9 staining co-localize with GABAergic and glutamatergic neurons.

<p>Fluorescence immunohistochemistry (a-d) on mouse brain paraffin embedded sections with SLC38A9 immunostaining in red, protein markers in green and the nuclear marker DAPI in blue. All scale bars are 20μm. White arrows indicate cells with SLC38A9 immunoreactivity and yellow arrows indicate cells with immunoreactivity of the markers. (a) The neuronal marker NeuN co-localizes in the 10th cerebellar lobule (10cb), Bregma -6.72mm. (b) The GABAergic neuronal marker GAD67 co-localized with SLC38A9 in cells close to third ventricle (3V), in anterior hypothalamic area, post (AHP), Bregma -1.22mm. (c) SLC38A9 and the astrocyte marker GFAP do not overlap in the area around third ventricle (3V), reuniens thalamic nucleus (Re), Bregma -0.70mm. (d) The enzyme glutaminase is expressed in glutamatergic neurons and overlap with SLC38A9 in the area B9 serotonin cells (B9), Bregma -4.36mm.</p

Western blot of the anti-SLC38A9 antibodies.

<p>Western blot was used to verify the antibodies. (a) The blot of the custom made anti-SLC38A9 antibody displayed a band with approximately size of 55kDa, which is ±10kDa of the predicted size of 63kDa of the SLC38A9 protein. (b) The blot of the commercial anti-SLC38A9 (HPA043785) antibody displayed a band with approximately size of 95kDa.</p