Figure 1P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . A - F . Clinical parameters determined in the study include age ( A ) , gender ( B ) , parasitemia ( C ) , hemoglobin ( g / dL blood ) ( D ) , hematocrit ( E ) , and platelet count ( F ) . Boxes represent the 25th to 75th percentiles , whiskers show the range ( minimum to maximum ) , and lines represent the median . Significance was determined by the Kruskal - Wallis test with Dunn ' s multiple comparisons ( A , D - F ) , the Chi - square test ( B ) , and the Mann - Whitney test ( C ) using 30 ( symptomatic ) , 40 ( asymptomatic ) and 31 ( healthy immune controls ) biological replicates . * * * * p < 0 . 0001 . P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . G . Antibody titers specific for P . falciparum parasite lysate . Bars represent the mean ± SD of using 29 ( symptomatic ) , 35 ( asymptomatic ) and 26 ( healthy immune controls ) biological replicates . The dotted line depicts the average antibody background levels of malaria - naive healthy Melbourne controls . H . Mean antibody titers as determined by ELISA to the P . falciparum recombinant proteins EBA - 175 , EBA - 140 , PfRh2 , PfRh4 , PfRh5 , and PfRipr .

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Figure 1P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age (A), gender (B), parasitemia (C), hemoglobin (g/dL blood) (D), hematocrit (E), and platelet count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. G. Antibody titers specific for P. falciparum parasite lysate. Bars represent the mean ± SD of using 29 (symptomatic), 35 (asymptomatic) and 26 (healthy immune controls) biological replicates. The dotted line depicts the average antibody background levels of malaria-naive healthy Melbourne controls. H. Mean antibody titers as determined by ELISA to the P. falciparum recombinant proteins EBA-175, EBA-140, PfRh2, PfRh4, PfRh5, and PfRipr.


Figure 2PBMCs from P . falciparum symptomatic ( n = 16 ) and asymptomatic ( n = 24 ) infected individuals as well as healthy immune controls ( n = 24 ) were stained with a panel of metal - labelled antibodies and analysed by CyTOF . tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub - populations within gated : A . TH1 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ) B . TH2 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ) C . Circulating memory TFH cells ( CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) D . Classical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ) E . Atypical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) F . Activated MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ) The tSNE plots in the top panel display cell density and represent the pooled data for each group , while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot . Heatmaps show the median marker expression for each FlowSOM cluster .

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Figure 2PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.


Figure 3Unsupervised identification of differentially abundant high - dimensional cell populations between clinical groups was performed using CITRUS ( FDR < 5 % ) . between P . falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls . A - E . tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers , relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3 + CCR6 - memory CD4 + T cells ( A ) , CXCR3 - CCR6 - memory CD4 + T cells ( B ) , circulating memory TFH cells ( C ) , classical MBCs ( D ) , and atypical MBCs ( E ) . The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays . The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay , whiskers showing the range ( minimum to maximum ) , with lines representing the median of 16 ( symptomatic ) , 24 ( asymptomatic ) and 24 ( healthy immune controls ) biological replicates , while the pink histograms illustrate marker expression in identified differentially abundant populations , relative to background expression , shown in lilac .

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Figure 3Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.


Figure 4PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . A . Estimated proportions of PBMC sub - populations determined from cell - type deconvolution from study participant transcriptional profiles . PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . D . Blood transcriptional module ( BTM ) analysis showing significant BTMs differentially enriched for pairwise comparisons ( FDR < 15 % ) .

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Figure 4PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. A. Estimated proportions of PBMC sub-populations determined from cell-type deconvolution from study participant transcriptional profiles.PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. D. Blood transcriptional module (BTM) analysis showing significant BTMs differentially enriched for pairwise comparisons (FDR<15%).


Figure 5Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . A . Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes ( DEGs ) between symptomatic P . falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance . B . Mean - difference plot displaying genes differentially expressed between symptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in symptomatic malaria . Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . F . Upstream regulator analysis of the 922 DEGs between symptomatic P . falciparum malaria and healthy immune controls . The red lines represent a significant activation z - score of ± 2 . Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . Hierarchical clustering heatmap ( G ) displaying DEGs in symptomatic P . falciparum malaria and healthy immune controls predicted to be controlled by IFN - γ and IL - 13 upstream regulators .

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Figure 5Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. A. Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes (DEGs) between symptomatic P. falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance. B. Mean-difference plot displaying genes differentially expressed between symptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in symptomatic malaria.Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. F. Upstream regulator analysis of the 922 DEGs between symptomatic P. falciparum malaria and healthy immune controls. The red lines represent a significant activation z-score of ±2.Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. Hierarchical clustering heatmap (G) displaying DEGs in symptomatic P. falciparum malaria and healthy immune controls predicted to be controlled by IFN-γ and IL-13 upstream regulators.


Figure 6Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . C . Heat map of the 418 DEGs in P . falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls . E . Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2 - fold - change and - Log10 ( P - value ) differentially expressed between P . falciparum symptomatic and asymptomatic infected individuals . Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria . Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . F . Mean RPKMs + SEM of selected genes in P . falciparum malaria symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) individuals . Dotted red lines depict transcriptional levels of healthy immune controls , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Figure 6Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria.Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05.


Figure 7Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . A . Hierarchical clustering heat map of the 171 differentially expressed genes ( DEGs ) in P . falciparum asymptomatic and healthy immune control participants . C . Mean - difference plot displaying DEGs between asyptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in asymptomatic malaria . Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . E . Mean RPKMs + SEM of selected genes in P . falciparum malaria asymptomatic individuals ( n = 5 ) and healthy immune controls ( n = 6 ) . Dotted red lines depict transcriptional levels of P . falciparum - infected symptomatic individuals , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Figure 7Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. E. Mean RPKMs + SEM of selected genes in P. falciparum malaria asymptomatic individuals (n=5) and healthy immune controls (n=6). Dotted red lines depict transcriptional levels of P. falciparum-infected symptomatic individuals, Mann-Whitney test of biological replicates, *p<0.05.


Figure 8A . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM + activated MBCs . B . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T - bet + CD4 + T cells .

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Figure 8A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells.


Figure 9B - G . PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 6 ) infected individuals as well as healthy immune controls ( n = 6 ) were stained with a panel of antibodies and analyzed by flow cytometry . tSNE plots ( B ) display cell density of the pooled data for each group , ( C ) shows a projection of the FlowSOM clusters on a tSNE plot , and ( D ) is a heatmap depicting the celluar phenotype of clusters of CTLA - 4 + CD3 + CD19 - cells . Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups ( E ) , Mann - Whitney test , * p < 0 . 05 . The panel on the left shows the average CTLA - 4 mean fluorescent intensity ( MFI ) or 6 biological replicates in cluster 4 + SEM , Mann - Whitney test , * * p < 0 . 01 , while representative histograms of CTLA - 4 expression in cluster 4 are shown on the right ( F ) . Marker intensity within clusters 1 and 6 ( G ) .

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Figure 9B-G. PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=6) infected individuals as well as healthy immune controls (n=6) were stained with a panel of antibodies and analyzed by flow cytometry. tSNE plots (B) display cell density of the pooled data for each group, (C) shows a projection of the FlowSOM clusters on a tSNE plot, and (D) is a heatmap depicting the celluar phenotype of clusters of CTLA-4+CD3+CD19- cells. Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups (E), Mann-Whitney test, *p<0.05. The panel on the left shows the average CTLA-4 mean fluorescent intensity (MFI) or 6 biological replicates in cluster 4 + SEM, Mann-Whitney test, **p<0.01, while representative histograms of CTLA-4 expression in cluster 4 are shown on the right (F). Marker intensity within clusters 1 and 6 (G).


Figure 1B . DCC and netrin - 1 expression ( FACS analysis ) in B - cells lymphocytes isolated from DCC + / + and DCCmut / mut mice spleen and lymph nodes . Results are presented as expression ratios between means expression levels in mutant animals ( n = 3 ) and control ones ( n = 3 ) . No significant difference was observed between wild - type and mutant animals . C . DCCmut / mut mouse with splenomegaly and systemic lymphadenopathy . Left panel : black and white arrows indicate respectively the inguinal and mesenteric lymph nodes . Right panel : enlarged spleen in a lymphoma - bearing mutant mouse ( left ) as compared to control organ ( right ) . D . Incidence of lymphoid proliferations and lymphoma in DCC + / + ( n = 19 ) and DCCmut / mut ( n = 29 ) mice . Tumors classification was performed blinded to genotype according to anatomopathologists consensus ( 29 ) . * : p = 0 . 036 ; one - tailed Fisher ' s exact test . E . Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice . Sections from control spleen , low - grade FL and high - grade DLBCL were stained with hematoxylin - eosin - safran or with antibodies to B220 or Ki67 . w : white pulp , r : red pulp , cr : compressed red pulp .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.


Figure 2A - B . Analysis of DCC ( A ) and netrin - 1 ( B ) expression in a cohort of normal tonsils ( n = 9 ) , Germinal - Center - B - Cell - like - DLBCL ( n = 13 ) , Activated - B - Cell - like - DLBCL ( n = 18 ) and MCL ( n = 36 ) . Quantification of genes expression was performed by Q - RT - PCR , relatively to HPRT housekeeping gene . * * : p < 0 . 005 ; * * * * : p < 0 . 0001 ; two - sided Mann - Whitney U - test . C - D . Analysis of netrin - 1 expression in DLBCL ( C ) and MCL ( D ) by immunohistochemistry . C . 21 DLBCL patients biopsies were used to build a TMA . Normal tissues ( normal tonsils , reactive lymph nodes , muscle , liver ) are included ( the left - most spot , upper panel , corresponds to liver ) . Enlarged representative image of netrin - 1 expression in DLBCL is shown ( lower panel ) . D . Representative images of netrin - 1 expression in paraffin - embedded biopsies of MCL ( n = 9 ) . Five tumors were positive for netrin - 1 ( left panel ) and four were negative ( right panel ) .

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Figure 2A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).


Figure 3A . Expression of netrin - 1 and DCC in lymphoma cell lines . Quantification was performed by Q - RT - PCR in 21 lymphoma cell lines . HPRT housekeeping gene was used as a standardization control . Netrin - 1 and DCC levels are indicated as follow : − , not detectable ; + to + + + + , moderate to very high expression . ABC and GC - DLBCL cell lines are highlighted respectively in blue and green , MCL ones are in grey . Expression of netrin - 1 ( in green ) by immunofluorescence using netrin - 1 antibody on OCI - Ly3 and Granta - 519 cell lines is shown on the right panel . Nuclei are counterstained in blue by Hoechst . B . Caspase - 3 activity in Granta - 519 cells after transfection of scrambled siRNA ( si - Ctl ) or netrin - 1 siRNA ( si - netrin - 1 ) , with or without DCC siRNA ( si - DCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 03 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test . D . Effect of net - 1 mAb on Granta - 519 ( left panel ) and OCI - Ly3 ( right panel ) cell density . Results are means + / - std indexed to control of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 03 ; two - sided Mann - Whitney U - test . E . Effect of net - 1 mAb on induction of Granta - 519 cells apoptosis , detected by TUNEL staining . Left panel : representative images are shown . TUNEL positive cells are labelled in red . Nuclei are counterstained in blue by Hoechst staining . Right panel : quantification of one representative experiment out of three performed is exposed . Results are presented as percentage of TUNEL - positive cells per field . * : p = 0 . 04 ; two - sided Mann - Whitney U - test . F . Caspase - 3 activity in Granta - 519 cells treated with net - 1 mAb antibody or with an unrelated Ig - G1 antibody ( Ctl ) , with or without silencing of DCC via siRNA ( siDCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test . G - H . Effect of netrin - 1 interfering antibody net - 1 mAb on OCI - Ly3 ( G ) and Granta - 519 ( H ) xenograft tumors growth . Results are presented as means + / - 95 % CIs of tumor growth rates from the beginning of treatment . * : p = 0 . 02 ; * * : p = 0 . 01 ; $ : p = 0 . 007 ; $ $ : p = 0 . 002 ; * * * : p = 0 . 0002 ; * * * * : p < 0 . 0001 ; two - sided Student t - test ( Granta - 519 ) or Welch t - test ( OCI - Ly3 ) . I . Caspase - 3 activity in tumors from control ( n = 4 ) or netrin - 1 - interfering antibody ( n = 4 ) treated mice . Tumors resection was performed at the end of the treatment . Results are means + / - std of caspase - 3 activity . * : p = 0 . 02 ; two - sided Mann - Whitney test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.


Figure 2 ( B ) Volcano plot showing differentially expressed genes between WT ( WT1 and WT2 ) and patient ( V . 1 ( F1 ) , V . 5 ( F1 ) ) primary dermal fibroblasts . The vertical axis ( y - axis ) shows the - log10 P - value , whereas the horizontal axis ( x - axis ) displays the log2 fold change value . The red dots represent the upregulated transcripts ; the blue dots represent the downregulated transcripts . A total of 172 genes were found significantly dysregulated . TAPT1 , a gene located in the IBD region , appeared among the most significantly downregulated genes in the patients .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients.


Figure 4 ( B ) qPCR results using specific primers for TAPT1 and TAPT1 - AS1 in 3 WT ( WT1 , WT2 , V . 2 ( F1 ) ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts . TAPT1 mRNA is significantly reduced in all patients compared to WTs , whereas TAPT1 - AS1 transcript levels are unaffected . Fold change relative to V . 2 ( F1 ) is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) . ( C ) Western blot analysis of endogenous TAPT1 protein ( ~ 60 kDa ) using whole protein extracts from primary dermal fibroblasts from WT ( WT1 and WT2 ) , heterozygous ( IV . 3 ( F1 ) ) and homozygous ( V . 1 ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) individuals and two different commercial antibodies ( top : Sigma , HPA042567 ; bottom : Sigma , HPA048658 ) . Results show a complete absence of TAPT1 protein in patient samples . GAPDH was used as a loading control . ( D ) qPCR analysis of TAPT1 expression in 3 WT ( WT1 , WT2 , WT3 ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts treated with cycloheximide ( CHX ) . CHX was used to block nonsense mediated decay ( NMD ) . Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells . For each graph , fold change relative to non - treated condition is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) qPCR results using specific primers for TAPT1 and TAPT1-AS1 in 3 WT (WT1, WT2 , V.2 (F1)) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts. TAPT1 mRNA is significantly reduced in all patients compared to WTs, whereas TAPT1-AS1 transcript levels are unaffected. Fold change relative to V.2 (F1) is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).(C) Western blot analysis of endogenous TAPT1 protein (~60 kDa) using whole protein extracts from primary dermal fibroblasts from WT (WT1 and WT2), heterozygous (IV.3 (F1)) and homozygous (V.1 (F1), V.5 (F1) and IV.1 (F2)) individuals and two different commercial antibodies (top: Sigma, HPA042567; bottom: Sigma, HPA048658). Results show a complete absence of TAPT1 protein in patient samples. GAPDH was used as a loading control.(D) qPCR analysis of TAPT1 expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts treated with cycloheximide (CHX). CHX was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


Figure 5 ( B ) Western blot analyses for TAPT1 ( ~ 60 kDa ) using cytosolic , Mito / ER / Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs ( WT1 and WT2 ) and two patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . TAPT1 protein is highly enriched in the Mito / ER / Golgi fraction , and to a lower extent in the nuclear fraction . GAPDH , TGN46 and BiP served as a cytosolic , Golgi network and ER markers , respectively . Adenylate Kinase ( AK2 ) was used as a mitochondrial marker . Laminin A / C was used as a nuclear marker . ( C ) Immunofluorescence staining of mitochondria using anti - TOM20 ( green ) , ER using anti - CANX ( red ) and Golgi using anti - GLG1 ( red ) in primary dermal fibroblasts from WT1 and V . 5 ( F1 ) . Similar staining patterns are observed with the three antibodies in both cell lines . Scale bar represents 10 µm . ( D ) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients ( - / - ) ( V . I ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) primary dermal fibroblasts compared to WTs ( + / + ) ( WT1 , WT2 and WT3 ) cells . Fold change relative to WT is plotted as mean ± SD . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) . ( E ) CMV cell infection assay on 2 patient ( V . 1 ( F1 ) and V . 5 ( F1 ) ) and 3 WT ( WT1 , WT4 and WT5 ) primary dermal fibroblast cell lines , using β - galactosidase activity as a readout . MRC5 cell line was used as a positive control . All of the cells were infected by the HCMV strain RC256 at a MOI = 0 . 1 . Data are shown as mean ± SD of three technical replicates . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Western blot analyses for TAPT1 (~60 kDa) using cytosolic, Mito/ER/Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs (WT1 and WT2) and two patients (V.5 (F1) and IV.1 (F2)). TAPT1 protein is highly enriched in the Mito/ER/Golgi fraction, and to a lower extent in the nuclear fraction. GAPDH, TGN46 and BiP served as a cytosolic, Golgi network and ER markers, respectively. Adenylate Kinase (AK2) was used as a mitochondrial marker. Laminin A/C was used as a nuclear marker.(C) Immunofluorescence staining of mitochondria using anti-TOM20 (green), ER using anti-CANX (red) and Golgi using anti-GLG1 (red) in primary dermal fibroblasts from WT1 and V.5 (F1). Similar staining patterns are observed with the three antibodies in both cell lines. Scale bar represents 10 µm.(D) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients (-/-) (V.I (F1), V.5 (F1) and IV.1 (F2)) primary dermal fibroblasts compared to WTs (+/+) (WT1, WT2 and WT3) cells. Fold change relative to WT is plotted as mean ± SD. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).(E) CMV cell infection assay on 2 patient (V.1 (F1) and V.5 (F1)) and 3 WT (WT1, WT4 and WT5) primary dermal fibroblast cell lines, using β-galactosidase activity as a readout. MRC5 cell line was used as a positive control. All of the cells were infected by the HCMV strain RC256 at a MOI=0.1. Data are shown as mean ± SD of three technical replicates. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


Figure 6 ( A ) Volcano plot showing differentially expressed genes as determined by RNA - seq in patient primary dermal fibroblasts ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) cells . The y - axis shows the - log10 P - value , whereas the x - axis displays the log2 fold change value . The red dots represent 75 significantly upregulated genes , and the blue dots represent 97 significantly downregulated genes . ( B ) qPCR validation test for 3 top dysregulated genes ( RARRES2 , ZIC1 , and ZIC4 ) detected by RNA - seq . The analysis was performed on RNA samples independent from those sent for RNA - seq for 2 WTs ( WT1 and WT2 ) and 2 patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . Fold change relative to WT1 is plotted as mean ± SD of three technical replicates . Asterisks indicate statistical significance ( Student t - test ; * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ) . ( C ) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) primary fibroblast cells using SI - NET - seq . The y - axis shows the - Log10 P - value , whereas the x - axis indicates the log2 fold change value for the Pol II occupancy . The Pol II density is increased in 222 genes ( red dots ) , and decreased in 95 genes ( blue dots ) . The yellow dot represents TAPT1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Volcano plot showing differentially expressed genes as determined by RNA-seq in patient primary dermal fibroblasts (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) cells. The y-axis shows the -log10 P-value, whereas the x-axis displays the log2 fold change value. The red dots represent 75 significantly upregulated genes, and the blue dots represent 97 significantly downregulated genes.(B) qPCR validation test for 3 top dysregulated genes (RARRES2, ZIC1, and ZIC4) detected by RNA-seq. The analysis was performed on RNA samples independent from those sent for RNA-seq for 2 WTs (WT1 and WT2) and 2 patients (V.5 (F1) and IV.1 (F2)). Fold change relative to WT1 is plotted as mean ± SD of three technical replicates. Asterisks indicate statistical significance (Student t-test; *** p-value < 0.001, **** p-value < 0.0001).(C) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) primary fibroblast cells using SI-NET-seq. The y-axis shows the -Log10 P-value, whereas the x-axis indicates the log2 fold change value for the Pol II occupancy. The Pol II density is increased in 222 genes (red dots), and decreased in 95 genes (blue dots). The yellow dot represents TAPT1.


Figure 1Dynamics of histone modifications after an acclimatizing HS ( ACC ) or no HS ( NHS ) in Col - 0 wild type at HSP22 . 0 ( A ) and HSP70 ( B ) . Seedlings were subjected to ACC or a control treatment ( NHS ) 4 d after germination . At the indicated time points after the treatments , ChIP - qPCR was performed with antibodies against H3K9ac , H3K4me3 , H3K4me2 and H3 . Schematics show positions of regions analyzed . Amplicon positions relative to TSS are HSP22 . 0 : 1 , - 2570 bp ; 2 , + 235 bp . HSP70 : 1 , 4192 bp downstream of the 3 ' UTR ; 2 , + 47 bp . Data shown are averages over three biological replicates . Amplification values were normalized to input , H3 and 4 h NHS region 2 . The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2 . Squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively , Student ´ s t - test ) between ACC and NHS samples of the same time point . Error bars indicate SE .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at HSP22.0 (A) and HSP70 (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2 and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are HSP22.0: 1, -2570 bp; 2, +235 bp. HSP70: 1, 4192 bp downstream of the 3'UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively, Student´s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE.


Figure 2A . Transcript levels of the indicated HS - inducible genes after ACC in Col - 0 ( blue bars ) and hsfa2 ( orange bars ) as determined by quantitative Reverse Transcription - PCR ( qRT - PCR ) . Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ( [ GENE OF INTERESTACC x h / TUB6ACC x h ] / [ GENE OF INTERESTNHS x h / TUB6NHS x h ] ) . Note that the y - axis is on a log10 scale . Error bars show SE of at least three biological replicates . Asterisks show significant differences between the genotypes at the same time point based on Student ´ s t - test ( * : p < 0 . 05 ; * * : p < 0 . 01 ) . B . Immunoblotting of HSP21 and HSP101 in Col - 0 ( C ) and hsfa2 ( h ) at the indicated time points after ACC or NHS . TUBULIN was used as a loading control . M , marker .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and hsfa2 (orange bars) as determined by quantitative Reverse Transcription-PCR (qRT-PCR). Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ([GENE OF INTERESTACC x h/ TUB6ACC x h]/ [GENE OF INTERESTNHS x h/ TUB6NHS x h]). Note that the y-axis is on a log10 scale. Error bars show SE of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student´s t-test (*: p<0.05; **: p<0.01).B. Immunoblotting of HSP21 and HSP101 in Col-0 (C) and hsfa2 (h) at the indicated time points after ACC or NHS. TUBULIN was used as a loading control. M, marker.


Figure 3H3K4me3 ( A ) or H3K4me2 ( B ) levels after an acclimatizing HS ( ACC ) in Col - 0 and hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 and APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) and hsfa2 ( orange bars ) seedlings were subjected to ACC or no treatment ( NHS ) 4 d after germination . At the indicated time points after the treatment , ChIP - qPCR was performed with antibodies against H3K4me3 ( A ) , H3K4me2 ( B ) and H3 ( for normalization ) . Schematics show positions of regions analyzed ( grey bars , UTR ; black bar , exons ) . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of the TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of the TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input and H3 and the Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0 and HSP70 ) or region 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at the same time point and treatment ; squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively ) between ACC and NHS samples of the same time point and genotype , Student ´ s t - test . Error bars indicate SE .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR. Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (grey bars, UTR; black bar, exons). Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0 and HSP70) or region 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.


Figure 4Figure 4 : Histone H3K9ac profiles after ACC in Col - 0 and hsfa2 at HSP18 . 2 , HSP22 . 0 , HSP70 and APX2 as detected by ChIP - qPCR . Col - 0 ( blue bars ) and hsfa2 ( orange bars ) seedlings were subjected to ACC or control treatment ( NHS ) 4 d after germination . At the indicated time points after the treatments , ChIP - qPCR was performed with antibodies against H3K9ac or H3 . Schematics ( grey bars , UTR ; black bar , exons ) show positions of regions analyzed . Intergenic control region 1 is 3123 bp ( APX2 ) or 2570 bp ( HSP22 . 0 ) upstream of the TSS , or 5311 bp ( HSP18 . 2 ) or 6725 bp ( HSP70 ) downstream of the TSS , respectively . Data are averages over four biological replicates . Amplification values were normalized to input and H3 and the Col - 0 4h NHS region 2 ( HSP18 . 2 , HSP22 . 0and HSP70 ) or 3 ( for APX2 ) . * , p < 0 . 05 ; * * , p < 0 . 01 for differences between genotypes at the same time point and treatment ; squares and triangles within bars mark significant differences ( p < 0 . 01 and p < 0 . 05 , respectively ) between ACC and NHS samples of the same time point and genotype , Student ´ s t - test . Error bars indicate SE .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Figure 4: Histone H3K9ac profiles after ACC in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR.Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac or H3. Schematics (grey bars, UTR; black bar, exons) show positions of regions analyzed. Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0and HSP70) or 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.


Figure 5Figure 5 : HSFA2 associates with HS memory - related genes transiently after an acclimatizing HS . Binding of HSFA2 - YFP to the promoter regions of HSP18 . 2 ( A ) , HSP22 . 0 ( B ) , APX2 ( C ) , HSP70 ( D ) and ACTIN7 ( E ) was determined by ChIP - qPCR . Quantifications represent the enrichment relative to input in % . Red - orange - yellow bars represent time points ( 0 . 5 h - 4 h - 28 h ) after a ACC and grey bars represent the respective NHS control samples . Schematics show the promoter region of the respective gene with regions analyzed ( arrow , TSS ; grey bar , 5 ' UTR ; black bar , exon ; black boxes , heat shock elements ( 5 ' - nnGnAnnTnCtn - 3 ' ) . Regions are drawn to scale . Intergenic control region 1 is 4610 bp ( HSP18 . 2 ) and 4192 bp ( HSP70 ) downstream of the 3 ' UTR and 2570 bp ( HSP22 . 0 ) or 3123 bp ( APX2 ) upstream of the TSS , respectively . One representative experiment out of at least four biological replicates is shown . Error bars are SD of three replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Figure 5: HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Binding of HSFA2-YFP to the promoter regions of HSP18.2 (A), HSP22.0 (B), APX2 (C), HSP70 (D) and ACTIN7 (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5'UTR; black bar, exon; black boxes, heat shock elements (5'-nnGnAnnTnCtn-3'). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (HSP18.2) and 4192 bp (HSP70) downstream of the 3'UTR and 2570 bp (HSP22.0) or 3123 bp (APX2) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.


Figure 6Figure 6 : Expression profiles of HS memory - related genes after a recurring HS show HSFA2 - dependent transcriptional memoryResponse of selected HS - inducible loci upon recurring HS as determined by qRT - PCR . Transcript levels of APX2 ( A ) , HSP18 . 2 ( B ) , HSP22 . 0 ( C ) , and HSP70 ( D ) in Col - 0 ( blue bars ) and hsfa2 ( orange bars ) at the indicated time points after a single HS or two HS separated by 2 d . Plants were subjected to 37 ° C for 60 min on either d 4 , d 6 or on d 4 + d 6 after germination . NHS samples were harvested at the same time points as HS d 4 samples . Transcript levels of four biological replicates normalized to TUB6 are shown . Small brackets compare the two genotypes at the same condition , large brackets compare the same genotype across two treatments ( at 1 h after treatment ) with Col - 0 indicated first ( Col - 0 / hsfa2 ) . Error bars show SE over four biological replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ´ s t - test .

Figure 6Figure 6: Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memoryResponse of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of APX2 (A), HSP18.2 (B), HSP22.0 (C), and HSP70 (D) in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to TUB6 are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ hsfa2). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.


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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O

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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Figure 6 ( 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 . ( 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 ) . 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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). 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.(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.


Figure 7 ( 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 . Data information : Data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 . ( 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 . Data information : Data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 . ( D ) The effect of ATF4 on the activity of the WT - UHMK1 promoter and its mutations , as evaluated by using luciferase reporter assays . Data information : Data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.(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. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.(D) The effect of ATF4 on the activity of the WT-UHMK1 promoter and its mutations, as evaluated by using luciferase reporter assays. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.(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.


Figure 8 ( 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 . Data information : Unpaired , two - tailed statistical significance was assessed by Student ' s t ‐ test . Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , . ( B ) Immunoblotting of UHMK1 , NCOA3 phosphorylated at S1062 / T1067 , NCOA3 , and ATF4 in the samples ( 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 . Data information : Unpaired , two - tailed statistical significance was assessed by Student ' s t ‐ test . Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , . ( D ) Immunoblotting of UHMK1 , NCOA3 phosphorylated at S1062 / T1067 , NCOA3 , and ATF4 in the samples ( 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 , .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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. Data information: Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data represent mean ± SD. *P< 0.05, **P< 0.01,.(B) Immunoblotting of UHMK1, NCOA3 phosphorylated at S1062/T1067, NCOA3, and ATF4 in the samples(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. Data information: Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data represent mean ± SD. *P< 0.05, **P< 0.01,.(D) Immunoblotting of UHMK1, NCOA3 phosphorylated at S1062/T1067, NCOA3, and ATF4 in the samples (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,.


Figure 9 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(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.