EMBO/soda-nel huggingface dataset:
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 2

Figure 2. DRD2 downregulation is mediated by lysosomal degradation. A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment. B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190. C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461). Values are reported as mean ± SEM of the ratio of protein vs. PSD-95 intensity level, normalized to the mean value of the Tor1a+/+ samples measured in the same experiment.
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 3

Figure 3. DRD2 down-regulation is mediated by enhanced degradative trafficking and mimicked by RGS9-2 silencing. WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151). C) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432). D) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087). E) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574). F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012). G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232). The graphs in A),B),D),E),G) report mean ± SEM of the ratio of protein vs. loading control intensity level, normalized to the Tor1a+/+ controls of the same experiment.
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 4

Figure 4. Increased p62 immunolabelling in striatal ChIs. Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008).
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 5

Figure 5. The AAV2/9-mCherry-GFP-LC3 reporter indicates upregulation of the autophagy pathway in ChIs. A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334). C) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM.
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 6

Figure 6. Subcellular localization of DRD2 in striatal ChIs. A) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets.
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 7

Figure 7. In vivo viral-mediated delivery of RGS9-2 into Tor1a+/- dorsal striatum rescues DRD2 protein level. A) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µm. B) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380.
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 8

Figure 8. RGS9-2 rescues DRD2 function in striatal ChIs. A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected area. B) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001). C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009). D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136).

Expanded View Figure Legends
Nucleotide degradation and ribose salvage in yeast (2013)
Figure 3

In carbon starvation, nucleosides and PPP compounds are produced by RNA degradation via autophagy in response to kinase signals. (A) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U-13C-glucose for 70?min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC-MS. (B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild-type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates). (C) Ratio of metabolite levels in atg7 strain versus wild-type strain in carbon starvation. (D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild-type strain in carbon starvation. (E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild-type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.
Nucleotide degradation and ribose salvage in yeast (2013)
Figure 4

Phm8 is the physiological yeast nucleotidase. (A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild-type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point. (B) Summary of transcripts data of PHM8, SDT1 and other enzymes in the pathway (Gasch et al, 2000; Bradley et al, 2009; Klosinska et al, 2011). All values are log2 transformed fold changes. (C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5?mM substrate and 5?mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1?µmol/mg/min are shown. The x axis represents specific phosphatase activity (µmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates). (D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (µmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).
Nucleotide degradation and ribose salvage in yeast (2013)
Figure 6

Nucleoside degradation causes S7P accumulation due to depletion of the other transaldolase substrate glyceraldehyde-3-phosphate, and SBP accumulation due to phosphorylation of S7P into SBP by Pfk1. (A) Diagram of proposed reactions. (BE) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild-type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).
Nucleotide degradation and ribose salvage in yeast (2013)
Figure 7

The ribose-salvage pathway is essential for yeast's survival in starvation. (A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates). (B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation. (D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).
Nucleotide degradation and ribose salvage in yeast (2013)
Figure 8

The accumulation of S7P in carbon starvation facilitates survival of subsequent oxidative stress. (A) Experimental design. Yeast cells growing on glucose were switched to no carbon media for 30?min. Thereafter, 20?mM H2O2 was added and metabolome analyzed by LC-MS. (B) Diagram of proposed reactions and their regulation. S7P accumulates during carbon starvation due to low level of glyceraldehyde-3-phosphate (GAP). Oxidative stress blocks GAP consumption via glycolysis by inhibiting GAPDH. This provides sufficient GAP to react with S7P, producing F6P via transaldolase. F6P is further converted to G6P which enters the oxidative PPP. The resulting NADPH is utilized for regeneration of reduced glutathione. 1,3-DPG, 1,3-diphosphoglycerate; GSSG, glutathione disulfide; GSH, reduced glutathione. (C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates). (E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 1

Figure 1. High expression of UHMK1 predicts poor prognosis of GC patients.

(A) UHMK1 levels in tumor tissues (T) and corresponding nontumor tissues (N) from five GC patients were analyzed using Western blotting and qRT-PCR. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test.

(B) Immunoblotting and qRT-PCR were used to measure the levels of UHMK1 in GES-1 and GC cell lines. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test.

(C) IHC analysis of UHMK1 expression in a GC patient tissue array (n=102). Representative images are shown. Scale bars=100μm.

(D) The IHC signals were scored using the Allred score as described in Material and Methods. Data were shown as mean ± standard deviation from three independent experiments.

(E) UHMK1 expression in 100 GC patients was analyzed based on the following parameters: tumor stage, tumor status, and lymph node invasion status. Fisher's test was applied to calculate the exact P value.

(F) Patients with GC were stratified by the UHMK1 level (n=100); OS and DFS were assessed by Kaplan-Meier analysis.

Data information: *P< 0.05, **P< 0.01, ***P< 0.001.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 2

Figure 2. UHMK1 knockdown strongly suppresses the proliferative and invasive abilities of GC cells, possibly via metabolic pathway reprogramming.

(A-C) (A) GC cells (SGC7901 and MGC803) were transfected with (A) shUHMK1-#1,-#2, -#3, or control shRNA lentiviral vector, or (B) with a shRNA-resistant expression construct, UHMK1Δ. Western blotting was used to measure the levels of UHMK1. NIH ImageJ software was used to quantify the band intensity. Western blotting assay were conducted for three replicates. (B and C) SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1Δ. CCK-8 and colony formation assays were conducted. Scale bars=5mm.

(D and E) The volumes of subcutaneous gastric tumors derived from SGC7901 cells in NOD/SCID mice were determined at different time points. Tumors and representative bioluminescence images are also shown. UHMK1 silencing in the mouse model was confirmed by western blotting (three biological replicates). Data were presented as mean ± standard deviation from shcontrol and shUHMK1#1 mice (n = 10 mice/group). Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Scale bars=1cm..

(F, G) UHMK1 silencing (F) and overexpression (G) affected GC cell abdominal metastasis. The quantitative number of metastatic nodules (left panel) in nude mice (n = 10 mice/group). Representative images of abdominal metastases are also shown (right panel). Scale bars=1cm.

(H) Heatmap of the top 500 genes upregulated in GC patients with high UHMK1 expression.

(I) Gene ontology enrichment analysis was used to analyze the top 20 enriched biological processes among GC patients with high UHMK1 expression.

Data information: **P< 0.01, ***P< 0.001, # marked no significance.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 3

Figure 3. UHMK1 upregulation significantly increases the proliferative and invasive abilities of GC cells by reprogramming purine metabolism. (A) Schematic representation of the main metabolic pathways.

(B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown. The data are shown in the heatmap.

(C) (upper panel) LC-MS/MS analysis was performed to measure 15N-glutamine-labeled purine synthesis intermediates in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs. (lower panel) LC-MS/MS was used to analyze metabolites labeled with 13C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs(three biological replicates).

(D) RNA and DNA with incorporated 14C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs were examined using LC-MS/MS(three biological replicates).

(E) qRT-PCR assays were used to analyze the effects of WT-UHMK1 or UHMK1-K54R on the genes controlling purine metabolism. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

(F) UHMK1 silencing significantly decreased SGC7901 cell proliferation, while ATIC overexpression or purine supplementation markedly reversed this inhibition. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. .

(G) Treatment with the ATIC inhibitor significantly reversed the proliferation of BGC823 cells induced by UHMK1 overexpression. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.