EMBO/soda-nel huggingface dataset:
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 4

Figure 4. ATF4 activation mediates UHMK1-enhanced purine biosynthesis and GC progression.

(A) (Ai) Western blot analysis was used to confirm UHMK1 overexpression and ATF4 knockdown in BGC823 cells. (Aii) Silencing ATF4 significantly decreased the levels of the indicated metabolites in BGC823 cells overexpressing UHMK1(three biological replicates). (Aiii) Silencing ATF4 significantly decreased the levels of RNA and DNA containing U-14C-glycine in BGC823 cells overexpressing UHMK1(three biological replicates).

(B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown or ATF4 reintroduction. The data are shown in the heatmap.

(C) Silencing ATF4 significantly decreased the levels of the indicated genes in BGC823 cells overexpressing UHMK1. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

(D) (Di) qRT-PCR was used to measure the expression of ATF4 in BGC823 cells overexpressing UHMK1. (Dii) UHMK1 overexpression enhanced the nuclear translocation of ATF4 in BGC823 cells. (Diii) ChIP assays on the ATIC and PPAT promoters were performed in BGC823 cells with the indicated antibody. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

(E) Silencing ATF4 markedly decreased the proliferation of BGC823 cells overexpressing UHMK1. (left panel) The quantitative number of colonies formed in scramble+Flag, scramble+Flag-UHMK1, siATF4+Flag and siATF4+Flag-UHMK1 group. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (right panel) Representative images of colonies are shown. Scale bars=5mm.

(F) Silencing ATF4 markedly decreased the migration of BGC823 cells with UHMK1 overexpression. (left panel) The quantitative number of migrated cells in scramble+Flag, scramble+Flag-UHMK1, siATF4+Flag and siATF4+Flag-UHMK1 group. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (right panel) Representative images of migrated cells in each group are shown. Scale bars=100μm.

(G) Silencing ATF4 markedly decreased the invasion of UHMK1 overexpressing BGC823 cells. (left panel) The quantitative number of invaded cells in scramble+Flag, scramble+Flag-UHMK1, siATF4+Flag and siATF4+Flag-UHMK1 group. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (right panel) Representative images of invaded cells in each group are shown. Scale bars=100μm.

Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 5

Figure 5. UHMK1 interacts with and phosphorylates NCOA3 at S1062/T1067.

(A) GST-UHMK1 full-length (FL), GST-UHMK1 ΔN (N-terminal truncated, 1-310aa), and GST-UHMK1 ΔC (C-terminal truncated, 311-419aa) were constructed (upper panel). GST labeled FL, ΔN and ΔC protein fragments were incubated with His-NCOA3. Pull-down, Coomassie blue staining, and Western blotting were performed (lower panel).

(B) GST-NCOA3 full-length (FL), GST-NCOA3 ΔN (1-408aa), GST-NCOA3 ΔS/T (409-921aa), GST-NCOA3 CBP (922-1161aa), and GST-NCOA3 ΔC (1162-1424aa) were constructed (upper panel).Pulldown assay with GST-NCOA3 FL or its protein fragments (ΔN, ΔS/T, CBP and ΔC) and His-UHMK1 were performed (lower panel).

(C) We performed an in vitro kinase assay by incubating purified NCOA3 FL or its protein fragments with purified UHMK1 kinase. These proteins were visualized using Coomassie blue staining. The phosphorylation of the substrates is shown in the autoradiograph.

(D) WT-UHMK1 or kinase-dead UHMK1-K54R protein was mixed with GST-CBP-WT or the indicated mutant, and in vitro kinase assays were performed.

(E) Silencing UHMK1 in SGC7901 cells via UHMK1-siRNA. NCOA3 phosphorylation at S1062/T1067 was measured with a special Ab that recognizes phosphorylated NCOA3-S1062/T1067.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 6

Figure 6. UHMK1-dependent phosphorylation of NCOA3 at S1062/T1067 promotes GC progression by promoting the de novo purine synthesis pathway.

(A) Knockdown of UHMK1 blocked NCOA3 binding to ATF4. SGC7901 cells were treated as indicated. Co-IP was carried out with mouse immunoglobulin G (IgG) or anti-NCOA3 antibody. Then Western blotting was performed.

(B) BGC823 cells were transfected with Flag-NCOA3 or NCOA3-S1062A/T1067A with or without His-tagged UHMK1. Co-IP was performed using an anti-Flag antibody.

(C) Proximity ligation assay of the interaction between different NCOA3 proteins and ATF4 in BGC823 cells. Confocal images were acquired with a Nikon A1 microscope and the Nikon Elements software suite. Maximum projection images are shown (original magnification ×120). Scale bars=20μm.

(D) Computational molecular docking analysis to investigate the molecular mechanism by which NCOA3-S1062/T1067 phosphorylation is involved in the interaction of NCOA3 binding to ATF4. Based on the predictions of ZDOCK software, S1062 and T1067 of NCOA3 are located in the interface between ATF4 and NCOA3, and S1062/T1067 phosphorylation greatly enhances the binding affinity of NCOA3 for ATF4 by increasing NCOA3 hydrophilicity.

(E) (Ei) ChIP data from BGC823 cells are presented as indicated. (Eii) NCOA3-S1062/T1067 phosphorylation by UHMK1 enhanced the expression of critical genes as indicated (three biological replicates).

(F) (Fi and Fii) The effects of WT-NCOA3 and the NCOA3-S1062A/T1067A, and NCOA3-S1062E/T1067E mutants on BGC823 cell proliferation, invasion, and migration (three biological replicates).

(G, H) The effects of WT-NCOA3, and the NCOA3-S1062A/T1067A, and NCOA3-S1062E/T1067E mutants on GC growth and metastasis in the mouse model (n = 10 mice/group). Scale bars=1cm.

Data information: Unpaired, two-tailed statistical significance was assessed by Student's t‐test.Data represent mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 7

Figure 7. ATF4 directly activates the transcription of the UHMK1 promoter in GC.

(A) SGC7901 and MGC803 cells were transfected with ATF4 shRNA or shcontrol. Western blottings and qRT-PCR were used to analyze the expression of UHMK1. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

(B) Overexpression of ATF4 enhanced the expression of UHMK1 in BGC823 and HGC27 cells. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test.

(C) The nucleotide sequences showing the -1348/-948 region in the human UHMK1 gene. The red text indicates the candidate ATF4 binding sites.

(D) The effect of ATF4 on the activity of the WT-UHMK1 promoter and its mutations, as evaluated by using luciferase reporter assays.

(E) ChIP assays were used to examine the binding of ATF4 to the UHMK1 promoter in SGC7901 and MGC803 cells. Input DNA and nonspecific IgG were included as controls.

(F) ATF4 deficiency and overexpression regulated the activity of the UHMK1 promoter in GC cells.

Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 8

Figure 8. H. pylori infection and GC-associated UHMK1 mutation significantly enhance UHMK1 activity, NCOA3 phosphorylation at S1062/T1067, and ATF4 activity in GC.

(A) qRT-PCR was performed to analyze UHMK1 expression in BGC823 cells infected with or without H. pylori CagA+ strains 7.13 and J166. MOI, multiplicity of infection. Data were presented as mean ± standard deviation from three replicates.

(B) Immunoblotting of UHMK1, NCOA3 phosphorylated at S1062/T1067, NCOA3, and ATF4 in the samples from (A).

(C) PMSS1, a H. pylori strain, was used to challenge mice orogastrically, with Brucella broth as the control. qRT-PCR was performed to measure UHMK1 (Ci) and PMSS1(Cii) expression in gastric tissues. Data were presented as mean ± standard deviation from three replicates.

(D) Immunoblotting of UHMK1, NCOA3 phosphorylated at S1062/T1067, NCOA3, and ATF4 in the samples from (C).

(E) An ATF4 luciferase reporter assay was carried out in BGC823 cells with or without H. pylori infection. Data were presented as mean ± standard deviation from three replicates.

(F) (Fi) The domain architecture of UHMK1 and the positions of human GC-associated mutations. (Fii and Fiii) IP kinase assay. Briefly, UHMK1 from HEK293 cells expressing WT-UHMK1 or its mutants was immunoprecipitated with anti-HA antibodies. The immunoprecipitated proteins were mixed with a synthetic peptide of the CATS protein (a known substrate of UHMK1) and [32P] ATP. A phosphocellulose paper assay was used to measure kinase activity. The results were normalized to a value of 1.0 for WT-UHMK1(three biological replicates).

(G) The effects of human GC-associated UHMK1 mutations on the proliferation and colony formation of GC cells (three biological replicates).

(H) The effects of human GC-associated UHMK1 mutations on GC cell invasion and migration (three biological replicates).

Data information: Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data represent mean ± SD. *P< 0.05, **P< 0.01,.
UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism (2019)
Figure 9

Figure 9. Clinical correlations of UHMK1 with p-NCOA3 (S1062/T1067) and ATIC in human GC samples.

(A) Images showing UHMK1, p-NCOA3 (S1062/T1067), and ATIC protein expression in two human GC specimens. Scale bars=100μm.

(B) Pearson correlation analysis about of the levels of UHMK1, p-NCOA3 (S1062/T1067) and ATIC proteins in patients with GC (n=120) by IHC.

(C) (Ci)OS data from GC patients stratified by the level of UHMK1. (Cii)Combination of UHMK1 and p-NCOA3 (S1062/T1067) expression predicts more exact OS of GC patients. Survival curves were plotted according to Kaplan-Meier method, with the log-rank test applied for comparison.

(D) Schematic depicting the function of UHMK1 in GC. H. pylori-induced upregulation of UHMK1 or human GC-associated UHMK1 mutation enhances the level of NCOA3 phosphorylation at S1062/T1067; this event greatly increases the association of NCOA3 with ATF4, which activates the de novo purine biosynthesis pathway and promotes the development of GC. Interestingly, activated ATF4 transcriptionally upregulated UHMK1 expression.

Expanded View Figure Legends
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 1

Figure 1. Effect of GSK3 isoform knockdown on basal synaptic transmission.

A. Experimental scheme for B-H. Whole-cell patch-clamp recordings were made from nearby pairs of uninfected and infected neurons in CA1 of organotypic hippocampal slices infected with lentivirus carrying shRNAs against GSK3α or GSK3β. EPSCs were elicited with CA3 Schaffer collateral stimulation.

B. Representative traces for C-H, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and shRNA-expressing neurons (green traces).

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.

E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.

F, G. As C, D, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.

H. As E, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.

Data information: Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs (panels C, D, F, G).
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 2

Figure 2. shRNA knockdown of GSK3ɑ, but not GSK3β, blocks NMDAR-dependent LTD.

A. Experimental scheme for B-E. Whole-cell patch-clamp recordings were made from nearby pairs of uninfected and infected neurons in CA1 of organotypic hippocampal slices infected with lentivirus carrying shRNAs against GSK3α or GSK3β. EPSCs were elicited with CA3 Schaffer collateral stimulation, and low-frequency stimulation (LFS) was used to induce LTD.

B. Representative traces for C, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (uninfected) and GSK3α (blue) or GSK3β (orange) shRNA-expressing neurons. Error bars represent SEM.

D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.

E. Cumulative frequency distribution of the ESPC values plotted in D. Statistical significance was calculated according to the Kolmogorov-Smirnov test.
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 3

Figure 3. Effect of acute pharmacological inhibition of GSK3 on basal synaptic transmission.

A. Representative traces for B and C, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from inactive control-, GSK3α or GSK3β inhibitor-treated slices (all at 10 µM). Slices were incubated with each compound for 1 hour before recording, and during whole-cell recording itself. Compounds were also included in the internal solution of the recording pipette (all at 10 µM).

B. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from inactive control, GSK3α or GSK3β inhibitor-treated slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.

C. Cumulative frequency distribution of the AMPA/NMDA ratios plotted in B. Statistical significance was calculated using the Kolmogorov-Smirnov test.

D. Representative traces for E and F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from hippocampal slices treated with DMSO (vehicle control), AR-18 (10 µM), CHIR (1 µM) or SC79 (20 µM).

E, F. As B, C, for DMSO, AR-18, CHIR and SC79. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 4

Figure 4. Pharmacological inhibition of GSK3ɑ, but not GSK3β, blocks NMDAR-dependent LTD.

A. Experimental scheme for B-G. Whole-cell patch-clamp recordings were made from neurons in CA1 of organotypic hippocampal slices treated with inactive control, GSK3α or GSK3β inhibitor, or AR-18 (all at 10 µM). EPSCs were elicited with CA3 Schaffer collateral stimulation, and low-frequency stimulation (LFS) was used to induce LTD. For inactive control, GSK3α and GSK3β inhibitor compounds, the experiment was performed blind with respect to treatment group.

B. Representative traces for C, D, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM.

D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.

E. As B, for F and G.

F. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of mGluR-dependent LTD by PP-LFS (900 paired pulses, separated by 50 ms, at 1 Hz), in control (inactive control) and GSK3α inhibitor-treated neurons (blue). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Representative traces for synaptic responses before (dark trace) and after (light trace) LTD induction are shown above the time course. Error bars represent SEM.

G. Quantification of average synaptic depression from the last 5 minutes of the time course shown in F. Error bars represent SEM. No statistically significant difference was found, according to the Mann-Whitney U test.
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 5

Figure 5. Increased GSK3 activity induces synaptic depression.

A. Experimental scheme for B-F. Whole-cell patch-clamp recordings were made from nearby pairs of uninfected and infected neurons in CA1 of organotypic hippocampal slices expressing EGFP-GSK3α or EGFP-GSK3β fusion proteins. EPSCs were elicited with CA3 Schaffer collateral stimulation.

B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α- or EGFP-GSK3β -expressing neurons (green traces).

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in slices expressing EGFP-GSK3α (C) or EGFP-GSK3β (D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.

E, F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at +40 mV (NMDAR-mediated EPSCs) in slices expressing EGFP-GSK3α (E) or EGFP-GSK3β (F). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.

Data information: Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs.
GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring (2020)
Figure 6

Figure 6. GSK3ɑ, but not GSK3β, is transiently anchored in dendritic spines during LTD.

A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm.

B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines).

C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm.

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test. N = 12, 11 and 11 spines for EGFP-GSK3α baseline, cLTD and washout groups and 20, 11 and 9 spines for EGFP-GSK3β baseline, cLTD and washout groups, respectively.

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α or EGFP-GSK3β in 'baseline', 'cLTD' or 'recovery' conditions. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test.

G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1.

H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01.