EMBO/SourceData huggingface dataset:
Figure 5(A) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 NESmut (V238A/L242A) (green) and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling) and anti-rabbit Alexa Fluor 680. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(B) Representative images of COS7 cells transfected with GFP-COP1 WT (green), the indicated TRIB1-HA constructs (red), and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling), anti-HA (Sigma), anti-rabbit Alexa Fluor 680, and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (A) and (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; *p < 0.01; ****p < 0.00001).(E) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(F) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (E) and Figure EV6C. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; ****p < 0.00001).(G) Quantification of percentage of cells observed in (E) and Figure EV6C with the indicated GFP-COP1 construct exhibiting nuclear (Nuc), nuclear and cytoplasmic (Nuc/Cyt), or cytoplasmic (Cyt) localization. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed.
Figure 1B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).
Figure 2B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.
Figure 3A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.
Figure 4Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.
Figure 5A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.
Figure 6Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.
Figure 7Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.
Figure 1(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.(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 Data were shown as mean ± standard deviation from three independent experiments. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.(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.
Figure 2A) 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. SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1Δ. CCK-8 assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1 colony formation assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.(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.. Data information: **P< 0.01, ***P< 0.001, # marked no significance.(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. Data information: **P< 0.01, ***P< 0.001, # marked no significance.(H) Heatmap of the top 500 genes upregulated in GC patients with high UHMK1 expression.
Figure 3(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). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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. . Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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.
Figure 4(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.(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.
Figure 5(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.