( 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 ) .

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(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 .

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(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.


Dynamics 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 .

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Dynamics 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.


A . 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 ) .

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A. 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 .

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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.


H3K4me3 ( 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 .

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H3K4me3 (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 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 .

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Figure 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 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 .

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Figure 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 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 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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


A ) GC cells ( SGC7901 and MGC803 ) were transfected with ( A ) shUHMK1 - # 1 , - # 2 , - # 3 , or control shRNA lentiviral vector , or ( B ) with a shRNA - resistant expression construct , UHMK1Δ . Western blotting was used to measure the levels of UHMK1 . NIH ImageJ software was used to quantify the band intensity . Western blotting assay were conducted for three replicates . 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 .

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A) GC cells (SGC7901 and MGC803) were transfected with (A) shUHMK1-#1,-#2, -#3, or control shRNA lentiviral vector, or (B) with a shRNA-resistant expression construct, UHMK1Δ. Western blotting was used to measure the levels of UHMK1. NIH ImageJ software was used to quantify the band intensity. Western blotting assay were conducted for three replicates. 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 .

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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 .

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(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 .

(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 .

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


( 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 .

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(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 .

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(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 .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

(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 .

O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

(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.


( 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 , .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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

(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 , .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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,.


( 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 .

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(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 .

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(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.


a , Immunoblot analysis of Atg5 and LC3 . Brain homogenates were prepared from six - week - old control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice . Immunoblot analysis was performed using antibodies against Atg5 and LC3 . GSK - 3β was used as a loading control . The positions of the Atg12 - Atg5 conjugate , LC3 - I and LC3 - II ( LC3 - PE conjugate ) are indicated . Atg5 monomer was not detected in either lane ( data not shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a, Immunoblot analysis of Atg5 and LC3. Brain homogenates were prepared from six-week-old control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice. Immunoblot analysis was performed using antibodies against Atg5 and LC3. GSK-3β was used as a loading control. The positions of the Atg12-Atg5 conjugate, LC3-I and LC3-II (LC3-PE conjugate) are indicated. Atg5 monomer was not detected in either lane (data not shown).


b , A representative male control ( Atg5flox / + ; nestin - Cre ) and an Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) littermate at three weeks of age .

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b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age.


c , Left , paw placement records of eight - week - old mice . Right , stride lengths corrected for paw base widths ( stride / width ratio ) of Atg5flox / flox ; nestin - Cre and control ( Atg5flox / + ; nestin - Cre ) littermate mice . Values represent means ± s . d . of four mice . Asterisk , P 0 . 01 ( Students t - test ) .

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c, Left, paw placement records of eight-week-old mice. Right, stride lengths corrected for paw base widths (stride/width ratio) of Atg5flox/flox; nestin-Cre and control (Atg5flox/+; nestin-Cre) littermate mice. Values represent means ± s.d. of four mice. Asterisk, P 0.01 (Students t-test).


d , Abnormal limb - clasping of an Atg5flox / flox ; nestin - Cre mouse compared with a control mouse ( Atg5flox / + ; nestin - Cre ) when suspended by its tail .

d, Abnormal limb-clasping of an Atg5flox/flox; nestin-Cre mouse compared with a control mouse (Atg5flox/+; nestin-Cre) when suspended by its tail.


e , Rotarod testing of Atg5flox / + ; nestin - Cre ( open symbols ) and Atg5flox / flox ; nestin - Cre ( closed symbols ) mice . One male and one female mouse were analysed for each genotype . The time until drop from the rod ( rotating at 20 r . p . m . ) is shown .

e, Rotarod testing of Atg5flox/+; nestin-Cre (open symbols) and Atg5flox/flox; nestin-Cre (closed symbols) mice. One male and one female mouse were analysed for each genotype. The time until drop from the rod (rotating at 20 r.p.m.) is shown.


a , HE - stained sections of the cerebral cortex , the gracile nucleus and cerebellum from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at three months of age . Purkinje cells are indicated with arrowheads . Arrows indicate eosinophilic spheroids , which represent axon swelling . Scale bar , 10 µm .

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a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age. Purkinje cells are indicated with arrowheads. Arrows indicate eosinophilic spheroids, which represent axon swelling. Scale bar, 10 µm.


b , Immunohistochemistry using an anti - calbindin antibody on cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre mice at six weeks of age . Scale bar , 25 µm .

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b, Immunohistochemistry using an anti-calbindin antibody on cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age. Scale bar, 25 µm.


c , Loss of Purkinje cells in Atg5flox / flox ; nestin - Cre mice . Purkinje cells were counted in comparable areas for each mouse , and three fields were counted in each area for each mouse . Data are normalized against values from control mice ( Atg5flox / + ; nestin - Cre ) . Values represent mean ± s . d . of three mice . Asterisk , P 0 . 01 ( t - test ) .

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c, Loss of Purkinje cells in Atg5flox/flox; nestin-Cre mice. Purkinje cells were counted in comparable areas for each mouse, and three fields were counted in each area for each mouse. Data are normalized against values from control mice (Atg5flox/+; nestin-Cre). Values represent mean ± s.d. of three mice. Asterisk, P 0.01 (t-test).


d , Apoptotic death of granular cells . Cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre mice at six weeks of age were subjected to TUNEL staining . Nuclei were stained with Hoechst 33258 . Scale bar , 500 µm .

d, Apoptotic death of granular cells. Cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age were subjected to TUNEL staining. Nuclei were stained with Hoechst 33258. Scale bar, 500 µm.


a , Immunohistochemistry of brain sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at six weeks of age , stained with an anti - ubiquitin antibody ( 1B3 ) . Scale bar , 10 µm .

a, Immunohistochemistry of brain sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at six weeks of age, stained with an anti-ubiquitin antibody (1B3). Scale bar, 10 µm.


b , Ubiquitin - positive inclusions in neurons . Sections of medulla from Atg5flox / flox ; nestin - Cre mice at six weeks of age were stained with an antibody against NeuN ( a neuron - specific nuclear protein ) and ubiquitin . Nuclei were stained with Hoechst 33258 . Scale bar , 10 µm .

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b, Ubiquitin-positive inclusions in neurons. Sections of medulla from Atg5flox/flox; nestin-Cre mice at six weeks of age were stained with an antibody against NeuN (a neuron-specific nuclear protein) and ubiquitin. Nuclei were stained with Hoechst 33258. Scale bar, 10 µm.


c , Immunoelectron microscopy of ubiquitin - positive inclusion bodies . DRG neurons isolated from Atg5 - / - neonates were analysed by immunoelectron microscopy using an anti - ubiquitin antibody . Scale bars , 500 nm .

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c, Immunoelectron microscopy of ubiquitin-positive inclusion bodies. DRG neurons isolated from Atg5-/- neonates were analysed by immunoelectron microscopy using an anti-ubiquitin antibody. Scale bars, 500 nm.


a , Immunohistochemistry of DRG neurons from Atg5 + / + and Atg5 - / - mice at different developmental stages ( embryonic day ( E ) 13 . 5 , E15 . 5 or postnatal day ( P ) 0 ) , stained with an antibody directed against ubiquitin . Scale bar , 25 µm .

a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E)13.5, E15.5 or postnatal day (P)0), stained with an antibody directed against ubiquitin. Scale bar, 25 µm.


b , Accumulation of Triton - X - 100 - solublepolyubiquitinated proteins in the brains of Atg5flox / flox ; nestin - Cre mice . Brain homogenate prepared at the indicated times from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice were separated into Triton - X - 100 - soluble ( S ) and - insoluble ( P ) fractions and analysed by immunoblotting using anti - ubiquitin antibodies . Arrowhead indicates the stacking gel .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O 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, Accumulation of Triton-X-100-solublepolyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel.


c , Immunohistochemistry of liver sections from control ( Atg5flox / + ; CAG - Cre ) and Atg5flox / flox ; CAG - Cre ( Atg5flox / flox ) mice at four months of age , using an anti - ubiquitin antibody . Scale bar , 25 µm .

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c, Immunohistochemistry of liver sections from control (Atg5flox/+; CAG-Cre) and Atg5flox/flox; CAG-Cre (Atg5flox/flox) mice at four months of age, using an anti-ubiquitin antibody. Scale bar, 25 µm.


d , Immunohistochemistry of liver sections from six - week - old Atg5flox / flox ; Mx1 - Cre mice at the indicated time points after pIpC injection , using anti - ubiquitin antibodies . Arrowheads indicate ubiquitin - positive inclusion bodies . Scale bar , 25 µm .

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d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm.


e , Five thousand hepatocytes were randomly selected , and the number of cells with diffuse cytosolic ubiquitin signals , with ( black ) or without ( white ) inclusion bodies , were counted .

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e, Five thousand hepatocytes were randomly selected, and the number of cells with diffuse cytosolic ubiquitin signals, with (black) or without (white) inclusion bodies, were counted.


Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5 - GFP or GFP - ATG8 . ( a , b ) Control conditions : cytosolic signal associated with rare fluorescent puncta . ( c , d ) High increase of fluorescent puncta after 2 h of nutrient starvation . Note ring - like ATG5 structures in c . ( e , f ) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures . ( g , h ) Maximum intensity projections of vacuolar focal planes , allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A . Vacuolar ATG5 - GFP fluorescence is not detected ( g ) , while GFP - ATG8 signals are abundant ( h ) . An inset in g , corresponding to a cortical focal plane , shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment . Maximum intensity projections of 10 - μm - deep deconvolved stacks , of cortical ( a - f , and inset in g ) or vacuolar focal planes ( g , h ) . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.


( a ) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A , in nutrient starvation conditions . Autophagic bodies accumulation in the vacuole is clearly visible in wild - type seedlings , absent in the atg5 - 1 mutant and restored in atg5 - 1 seedlings expressing ATG5 - GFP . ( b ) Confocal microscopy . The ATG5 - GFP fluorescence pattern in the atg5 - 1 background is identical to that observed in wild - type seedling , including ring - like structures . Scale bar , 15 μm .

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(a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.


( a ) Dual observation of ATG5 - GFP and mCh - ATG8 under nutrient starvation reveals a partial colocalization of the two reporters . Merge of the GFP ( green ) and ATG8 ( magenta ) channels of maximum intensity projections of 20 - μm - deep deconvolved stacks . Scale bar , 3 μm .

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(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm.


( b ) Real - time imaging of an individual autophagosomal structure , showing one deconvolved image per 20 s . The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure . The following steps , defined as the decay phase , being more dynamic , are depicted also at higher time resolution . Single - plane merged images of ATG5 - GFP ( green ) and mCh - ATG8 ( magenta ) channels , deconvolved . Scale bar , 1 μm .

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(b) Real-time imaging of an individual autophagosomal structure, showing one deconvolved image per 20 s. The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure. The following steps, defined as the decay phase, being more dynamic, are depicted also at higher time resolution. Single-plane merged images of ATG5-GFP (green) and mCh-ATG8 (magenta) channels, deconvolved. Scale bar, 1 μm.


( c ) Representative FRAP experiment on mCh - ATG8 during the ring formation phase . Deconvolved images . Scale bar , 1 μm . The corresponding fluorescence recovery kinetic , measured on non - deconvolved images , is shown on the right .

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(c) Representative FRAP experiment on mCh-ATG8 during the ring formation phase. Deconvolved images. Scale bar, 1 μm. The corresponding fluorescence recovery kinetic, measured on non-deconvolved images, is shown on the right.


( d ) Representative FRAP experiment of mCh - ATG8 during the ring decay phase , presented as in c .

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(d) Representative FRAP experiment of mCh-ATG8 during the ring decay phase, presented as in c.


( e ) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation ( n = 11 ) and decay ( n = 17 ) . Whiskers comprise the 10 - 90 percentile , the box the two central quartiles . The mean recovery values are indicated by an horizontal line . Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann - Whitney statistical test ( * * * P0 . 001 ) .

(e) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation (n=11) and decay (n=17). Whiskers comprise the 10-90 percentile, the box the two central quartiles. The mean recovery values are indicated by an horizontal line. Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann-Whitney statistical test (***P0.001).


( a ) Correlative light - electron microscopy of GFP - ATG8 - labelled autophagosomal structures . Correlation of a TEM micrograph of a root transverse section ( 1 ) with fluorescent imaging of GFP - ATG8 ( arrowheads , 2 ) confirms the association of ATG8 signal with autophagosome ( 3 ) . A closer view shows close contacts with ER - like membranes ( empty arrowheads , 4 ) .

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(a) Correlative light-electron microscopy of GFP-ATG8-labelled autophagosomal structures. Correlation of a TEM micrograph of a root transverse section (1) with fluorescent imaging of GFP-ATG8 (arrowheads, 2) confirms the association of ATG8 signal with autophagosome (3). A closer view shows close contacts with ER-like membranes (empty arrowheads, 4).


( b ) Immunogold labelling of GFP - ATG8 on cryosections , employing an anti - GFP antibody ( arrowheads ) . Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed ( samples c ) or after chemical fixation ( d ) . Empty arrowheads : connections with ER - like membranes . Scale bars , 5 μm in a ( 1 - 3 ) ; 0 . 5 μm in a ( 4 ) and b ( 1 - 3 ) .

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(b) Immunogold labelling of GFP-ATG8 on cryosections, employing an anti-GFP antibody (arrowheads). Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed (samples c) or after chemical fixation (d). Empty arrowheads: connections with ER-like membranes. Scale bars, 5 μm in a(1-3); 0.5 μm in a(4) and b(1-3).


( A and B ) THP1 - derived macrophages were treated with the TLR ligands Pam3 , lipopolysaccharide ( LPS ) , or FSL - 1 and analyzed by RNA - seq 8 h later . Circos plots show genome - wide differential expression of protein - coding genes ( A ) and non - protein - coding genes ( B ) between untreated and treated macrophages . The outermost to innermost circles show downregulated ( blue ) or upregulated ( red ) genes in cells stimulated with FSL - 1 , LPS , and Pam3 , respectively . Chromosomes are indicated by the numbers 1 - 22 and X and Y .

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(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.


( F ) RT - qPCR validation of differentially expressed lncRNAs and mRNAs in THP1 - derived macrophages stimulated for 8 h with flagellin ( FLA ) , FSL - 1 , imiquimod , LPS , Pam3 , or poly ( I : C ) . Heat map shows the fold change in expression of 13 mRNAs ( left ) and lncRNAs ( right ) .

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(F) RT-qPCR validation of differentially expressed lncRNAs and mRNAs in THP1-derived macrophages stimulated for 8 h with flagellin (FLA), FSL-1, imiquimod, LPS, Pam3, or poly(I:C). Heat map shows the fold change in expression of 13 mRNAs (left) and lncRNAs (right).


( A ) Activation and suppression of nearby genes by eight cis - acting lncRNAs . RT - qPCR analysis of THP1 - derived macrophages transduced with lentiviruses expressing non - targeting control ( NC ) shRNA or shRNAs targeted to the indicated lncRNAs and protein - coding genes . Mean ± SD of n = 3 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by Student ' s t test .

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(A) Activation and suppression of nearby genes by eight cis-acting lncRNAs. RT-qPCR analysis of THP1-derived macrophages transduced with lentiviruses expressing non-targeting control (NC) shRNA or shRNAs targeted to the indicated lncRNAs and protein-coding genes. Mean ± SD of n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001 by Student's t test.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 IL - 6 ELISA quantification ( C ) of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . ELISA were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 IL-6 ELISA quantification (C) of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. ELISA were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 1α ( D ) . RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-1α (D). RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


lnc - MARCKS ( B ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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lnc-MARCKS (B) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


, MARCKS ( C ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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, MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


IL - 6 ( D ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


Overexpression of lnc - MARCKS decreases MARCKS expression RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . Lnc - MARCKS and MARCKS expression ( F )

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Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F)


Overexpression of lnc - MARCKS enhances IL - 6 expression . RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . IL - 6 expression ( G ) .

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Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect lnc - MARCKS ( H )

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H)


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS ( I ) ,

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I),


lnc - MARCKS regulates IL - 6 in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect IL - 6 expressions ( J ) .

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lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).


( A ) ROCKI is recruited to the MARCKS promoter in Pam3 - stimulated THP1 - derived macrophages . ChIRP analysis of THP1 - derived macrophages treated with Pam3 for 8 h . ChIRP assays were performed using a non - specific lacZ probe or probes specific for lnc - MARCKS . Specificity of ROCKI probes ( left ) , and enrichment at the MARCKS promoter ( right ) .

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(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right).


( B ) Mass spectrometry identifies APEX1 as a candidate ROCKI - binding protein in THP1 - derived macrophages . APEX1 MS / MS count represents the ratio between the number of APEX1 peptides and all peptides detected . Mean of n = 2 samples . ROCKI and ROCKI - AS represent ROCKI and antisense strand of ROCKI transcripts , respectively .

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(B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively.


( C ) ROCKI inhibition of MARCKS expression is dependent on APEX1 . RT - qPCR analysis of MARCKS , ROCKI , and APEX1 expression in THP1 - derived macrophages overexpressing ROCKI and / or depleted of APEX1 .

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(C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1.


( D ) APEX1 specially binds to ROCKI . Western blot analysis of APEX1 in lacZ , or ROCKI pull - down fractions . Vimentin , which non - specifically binds to all RNA probes , is shown as a loading control .

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(D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control.


( E ) ROCKI associates with APEX1 . Left : RT - qPCR analysis of ROCKI present in anti - APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3 - treated THP1 - derived macrophages . Right : APEX1 western blot of the same immunoprecipitates .

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(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.


ChIP analysis of THP1 - derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter . RT - qPCR analysis of fragments of the MARCKS promoter region ( a and b ) or a non - specific region ( nc ) co - immunoprecipitated with anti - APEX1 antibody .

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ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody.


ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA ( NC ) or ROCKI shRNA and treated with Pam3 for 8 h . The y - axis shows the percentage of enrichment in normalized to input . An unrelated region of the ACTB2 promoter was amplified as a control .

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ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control.


Left : Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti - APEX1 antibody immunoprecipitates from NC ( control ) or ROCKI knockdown THP1 - derived macrophages . Right : RT - qPCR analysis of ROCKI in the same immunoprecipitates .

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Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates.


Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter . ChIP - qPCR analysis of HDAC1 was performed using an anti - HDAC1 antibody .

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Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed using an anti- HDAC1 antibody.


Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter . ChIP - qPCR analysis of H3K27ac was performed using an anti - H3K27ac antibody .

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Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.


Heatmap of representative genes commonly upregulated in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages .

Heatmap of representative genes commonly upregulated in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages.


RT - qPCR validation of inflammatory gene expression in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages stimulated with Pam3 for 8 h . E . V . , empty vector ; NC , control shRNA .

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RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA.


MARCKS is a negative regulator of Ca2 + signaling . Fluo - 4 fluorescence of MARCKS , ROCKI , or NC ( control ) shRNA - expressing THP1 - derived macrophages before and after treatment with PMA and Pam3 . Triton X - 100 was added to obtain the maximal [ Ca2 + ] signal . Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence ( ΔF / Fmax ) .

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MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).


( A ) QTL signals for ROCKI ( LINC01268 ) gene expression in whole blood ( top , black ) , H3K27ac promoter signal in monocytes ( middle , green ) , and H3K4me1 signal in monocytes ( bottom , orange ) in a 400 kb window around the MARCKS / ROCKI locus . The signals were strongly co - localized ( Pshared = . 97 , Pshared = . 98 ) suggesting they represent the same signal .

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(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.


( C ) RT - qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M . tuberculosis - infected donors .

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(C) RT-qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M. tuberculosis-infected donors.


( B ) Southern Blot analysis of targeted ES cells . Positions of wild type ( WT - 14 . 7kb ) and targeted ( loxP - 12 . 8kb ) alleles are indicated .

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(B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated.


( C ) Genotyping analysis of wild type ( Clpp + / + ) , heterozygous ( Clpp + / - ) and homozygous ( Clpp - / - ) Clpp knockout animals . DNA fragments corresponding to the wild type ( WT - 199 bp ) , and knockout ( KO - 273 bp ) Clpploci are indicated .

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(C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpploci are indicated.


( D ) Analysis of Clpp transcript levels in heart ( n = 4 ) . Bars represent mean ± standard deviation ( SD ) . Student ' s t - test was used to determine the level of statistical difference .

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(D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student's t-test was used to determine the level of statistical difference.


( E ) Western blot analysis of CLPPprotein levels in heart ( He ) , quadriceps ( Quad ) , liver ( Li ) , kidney ( Ki ) , lung ( Lu ) , brain ( Br ) , spleen ( Sp ) and pancreas ( Pa ) . TUBULIN and ACTIN are used as a loading control . The arrowhead indicates positions of CLPP , and the star indicates position of cross - reacting bands .

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(E) Western blot analysis of CLPPprotein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.


( C ) Respiratory chain enzyme activities . Relative enzyme activities of respiratory chain enzyme C I ( NADH coenzyme Q reductase ) , C I / III ( NADH cytochrome c reductase ) , C II ( succinate dehydrogenase ) , C II / III ( succinate cytochrome c reductase ) and C IV ( cytochrome c oxidase ) in mitochondrial extracts from wild type and Clpp knockout hearts . ( n = 5 ) . Bars present mean levels ± SD . Statistical difference was calculated by Student ' s t - test and changes are presented in the figure ( n = 5 ) .

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(C) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student's t-test and changes are presented in the figure (n=5).


( D ) Substrate - dependent oxygen consumption rates in isolated heartmitochondria . Complex I substrates ( pyruvate , malate , glutamate ) ; complex II substrate ( succinate ) . State 3 - ADP stimulated oxygen consumption rate ; State 4 - respiration rate after addition of oligomycin ; Uncoupled - oxygen consumption rate in the presence of FCCP . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 7 ) .

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(D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7).


( E ) Decrease of steady - state levels of respiratory chain subunits in Clpp - knockout hearts relative to wild type ( set to 1 ) . VDAC or TOMM20 were used as loading controls . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) .

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(E) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=4).


( F ) Blue Native Page Analysis ( BN - PAGE ) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I ( anti - NDUFA9 ) ; C III ( anti - UQCRC1 ) and C IV ( anti - COX4A ) .

(F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).


( A ) Quantification of mtDNA levels after Southern blot analysis . Bars present mean levels ± SD ( n = 4 ) .

(A) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4).


( B - C ) Northern blot analysis and corresponding quantification of mitochondrial ( B ) rRNAs and mRNAs or ( C ) tRNAs . 18S rRNA is used as loading control . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) .

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(B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).


( D ) Western blot analysis of LRPPRC levels . VDAC was used as a loading control .

(D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control.


( E ) In organello transcription analysis upon 60 minutes labelling with - 32P - UTP .

(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.


( A ) De novo synthesized mitochondrial proteins were isolated after labelling with 35S - methionine for 1 hour . Positions of individual mitochondrial encoded proteins are indicated .

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(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.