Sequences and their shared identities from Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression

The EMS file 1 is combined of three sheets, where the first includes all sequences used in the analyses. The second sheet (Atypical SLC matric 1) includes all sequences comparisons between the atypical SLCs, where the percentage represent sequence identities. Those pairs of proteins sharing more than 20 % identities (grey) could potentially constitute an SLC family. The third sheet (MFS matrix) is also a matrix showing sequence identities between the atypical SLCs and known SLC proteins originating from the MFS Pfam clan. Sequences sharing at least 20 % sequence identities are marked in grey

Proteins related to the atypical SLCs from Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression

With Hidden Markov Models, proteins related to the human atypical SLCs were found. They were annotated and listed with corresponding accession numbers (CG numbers for hits found in fruit flies). Proteins found in proximity to the human output proteins were given the same name as the human correspondent, whereas more distant related proteins were assigned the “L” (Like) suffix to highlight the proteins closeness

Abbreviation list from Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression

Abbreviations included in the manuscript

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

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

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

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

SLC38A9 immunostaining is abundant in mouse brain using a commercial anti-SLC38A9 antibody.

<p>Additional DAB immunohistochemistry on free floating mouse brain sections was performed using a commercially available anti-SLC38A9 antibody, to verify the staining of the custom made anti-SLC38A9 antibody. Overview pictures (a-d) and close up pictures (e-k) with adjacent scale bars. (a) Bregma -0.10mm, (b) Bregma -0.70mm, (c) Bregma -1.94mm, (d) Bregma -5.88mm, (e) cingulate cortex, area 1 (Cg1), primary motor cortex (M1) and secondary motor cortex (M2), Bregma -0.10mm, (f) piriform cortex (Pir) and caudate putamen (striatum) (CPu), Bregma -0.10mm, (g) third ventricle (3V), suprachiasmatic nucleus (SCh) and anterior hypothalamic area, central (AHC), Bregma -0.70mm, (h) paraventricular thalamic nucleus (PVA), Bregma -0.70mm, (i) hippocampus, field CA1 hippocampus (CA1), field CA2 hippocampus (CA2), field CA3 hippocampus (CA3) and dentate gyrus (DG), Bregma -1.94mm, (j) gigantocellular reticular nucleus (Gi), Bregma -5.88mm, (k) Purkinje cell layer in simple lobule (Sim) and crus 1 ansiform lobule (Crus1), Bregma -5.88mm.</p

SLC38A9 immunostaining is abundant in mouse brain using a custom made anti-SLC38A9 antibody.

<p>Non-fluorescent DAB immunohistochemistry on free floating mouse brain sections, overview pictures (a-e) and close ups (f-n) with adjacent scale bars. (a) Bregma 0.26mm, (b) Bregma -0.82mm, (c) Bregma -1.58mm, (d) Bregma -3.08mm, (e) Bregma -5.88mm, (f) piriform cortex (Pir) and caudate putamen (striatum) (CPu), Bregma 0.26mm, (g) cingulate cortex, area 2 (Cg2), secondary motor cortex (M2) and primary motor cortex (M1), Bregma 0.26mm, (h) Third ventricle (3V), Suprachiasmatic nucleus (SCh) and anterior hy area, central (AHC), Bregma -0.82mm, (i) paraventricular thalamic nucleus anterior, (PVA), Bregma -0.82mm, (j) hippocampus, field CA1 hippocampus (CA1), field CA2 hippocampus (CA2), field CA3 hippocampus (CA3) and dentate gyrus (DG), Bregma -1.58mm, (k) superior Cb peduncle (scp), Bregma -3.08mm, (l) ventral tegmental area (VTA), Bregma -3.08mm, (m) Facial nucleus (7N), Bregma -5.88mm, (n) Purkinje cell layer in simple lobule (Sim) crus 1 ansiform lobule (Crus1), Bregma -5.88mm.</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