D . Surface biotinylation followed by western blot showing the surface expression of the PC1 _ T3041V / PC2 _ AA complex , but not the PC1 _ L3040H / PC2 _ AA complex , in Xenopus oocytes . PC2 _ AA is completely absent from the surface sample when PC1 _ L3040H was coexpressed , indicating that , in our experimental condition , almost all PC2 _ AA were in the complex with PC1 _ L3040H and trapped in the process of cell surface trafficking . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 , and 130 kDa PC2 ( star ) are indicated .

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D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.


( 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 .

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

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

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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(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 .

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(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 .

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


D Representative confocal images of HEK293 cells expressing various GFP - fused proteins ( green ) and supplemented with TopFluor ® TMR - PS ( red ) ; scale bar = 10 µm . Yellow arrows indicate high intensity PS fluorescent regions . E % PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D . Black bars are control proteins and blue bars are eVP40 proteins . Values are reported as mean ± s . d . ; N > 18 cells per biological replicate , n = 3 for 3 biological replicates ; A one - way ANOVA was performed with multiple comparisons compared to the control GFP % PS clustering ( * * * p = 0 . 0007 , * * p = 0 . 004 ) . Data information : PS : phosphatidylserine ; PM : plasma membrane .

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

D Representative confocal images of HEK293 cells expressing various GFP-fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar = 10 µm. Yellow arrows indicate high intensity PS fluorescent regions. E %PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D. Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N>18 cells per biological replicate, n=3 for 3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the control GFP %PS clustering (***p=0.0007, **p=0.004). Data information: PS: phosphatidylserine; PM: plasma membrane.


D , E PS concentration in LUVs enhances the ability of His6 - eVP40 to oligomerize on membranes . D Representative western blot of chemical crosslinking performed on His6 - WT - eVP40 following incubation with LUVs of varying PS content ( detected by Mouse α - His antibody & HRP - Sheep α - Mouse ) . E Oligomerization capacity was determined from the western blot band density ratio of oligomers / ( monomer + dimer ) from chemical crosslinking experiments . A one - way ANOVA was performed with multiple comparisons compared to the control 0 % PS LUVs control ( 30 % PS * p = 0 . 02 ; 60 % PS * p = 0 . 017 ) . n = 3 biological replicates . Values are reported as mean ± s . d . ; Data information : LUVs : large unilamellar vesicles ; PS : phosphatidylserine ; HRP : horseradish peroxidase .

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

D, E PS concentration in LUVs enhances the ability of His6-eVP40 to oligomerize on membranes. D Representative western blot of chemical crosslinking performed on His6-WT-eVP40 following incubation with LUVs of varying PS content (detected by Mouse α-His antibody & HRP-Sheep α-Mouse). E Oligomerization capacity was determined from the western blot band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs control (30% PS *p= 0.02; 60% PS *p=0.017). n=3 biological replicates. Values are reported as mean ± s.d.; Data information: LUVs: large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.


D , E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) imaging of HEK293 expressing GFP - LactC2 and treated with fendiline for 48 hours ; scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - 5mer , green : 5mer - 10mer , blue : > 10mer ) . E Average % pixels quantification from panel ( d ) = Percentage of GFP - LactC2 with brightness values corresponding to monomer - 5mer ( ~ 1 . - 1 . 5 ) , 5mer - 10mer ( ~ 1 . 5 - 1 . 9 ) and > 10mer ( > 1 . 9 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons comparted to the control DMSO % average pixels ( * * * * p < 0 . 0001 , * * p = 0 . 0043 ) . Data information : GFP - LactC2 : phosphatidylserine sensor ; N & B : Number & Brightness analysis ; PS : phosphatidylserine .

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

D, E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N&B analysis. D Left panel: Representative images from time-lapse (30 frames) imaging of HEK293 expressing GFP-LactC2 and treated with fendiline for 48 hours; scale bar= 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-5mer, green: 5mer-10mer, blue: >10mer). E Average % pixels quantification from panel (d)= Percentage of GFP-LactC2 with brightness values corresponding to monomer-5mer (~1.-1.5), 5mer-10mer (~1.5-1.9) and >10mer (>1.9) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons comparted to the control DMSO % average pixels (****p<0.0001, **p=0.0043). Data information: GFP-LactC2: phosphatidylserine sensor; N&B: Number & Brightness analysis; PS: phosphatidylserine.


Effect of fendiline on EBOV infection . A Representative confocal images of Vero E6 cells infected with EBOV ( Kikwit ) at the indicated MOI and treated with the indicated concentration of fendiline . Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline . Post infection , cells were treated 1 hour later ( d - 1 / 0 ) , treated every day ( e . d ) , or treated every other day ( e . o . d ) and fixed at either 48 hours , 72 hours or 96 hours post infection . ( green = EBOV ; blue = nuclei ) . White numbering in top right corner indicates % infection . Scale bar = 100 μm . Data information : EBOV : Ebola virus ; MOI : multiplicity of

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

Effect of fendiline on EBOV infection. A Representative confocal images of Vero E6 cells infected with EBOV (Kikwit) at the indicated MOI and treated with the indicated concentration of fendiline. Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline. Post infection, cells were treated 1 hour later (d -1/0), treated every day (e.d), or treated every other day (e.o.d) and fixed at either 48 hours, 72 hours or 96 hours post infection. (green=EBOV; blue= nuclei). White numbering in top right corner indicates %infection. Scale bar = 100 μm. Data information: EBOV: Ebola virus; MOI: multiplicity of infection


Effect of fendiline on EBOV infection . B - D Quantification of % inhibition of EBOV by fendiline . B 48 hours ( MOI 1 . 0 ) C 72 hours ( MOI 0 . 1 ) D 96 hours ( MOI 0 . 1 ) . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons was performed . n = 3 biological replicates . Data information : EBOV : Ebola virus ; MOI : multiplicity of infection ; d . - 1 / 0 : treatment 1 hour after infection ; e . d . : treatment every day ; e . o . d . : treatment every other day .

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

Effect of fendiline on EBOV infection. B-D Quantification of % inhibition of EBOV by fendiline. B 48 hours (MOI 1.0) C 72 hours (MOI 0.1) D 96 hours (MOI 0.1). Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons was performed. n=3 biological replicates. Data information: EBOV: Ebola virus; MOI: multiplicity of infection; d. -1/0: treatment 1 hour after infection; e.d.: treatment every day; e.o.d.: treatment every other day.


A - C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment . A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP - WT - eVP40 ( top panel ) and EGFP - WT - mVP40 ( bottom panel ) after 48 hours of fendiline treatment . scale bars = 10 µm . Effect of fendiline on mVP40 ( B ) and eVP40 ( C ) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity ( and normalized to DMSO control ) . N > 15 cell per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA with multiple comparisons was performed compared to the DMSO control . Data information : PM : plasma

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

A-C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment. A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40 (bottom panel) after 48 hours of fendiline treatment. scale bars= 10 µm. Effect of fendiline on mVP40 (B) and eVP40 (C) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity (and normalized to DMSO control). N>15 cell per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control. Data information: PM: plasma membrane


D , E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) of HEK293 expressing EGFP - WT - eVP40 and treated with fendiline for 48 hours . scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - hexamer , green : hexamer - 12mer , blue : 12mer - 24mer , pink : > 24mer ) . E Average % pixel quantification from panel ( d ) = % of GFP - WT - eVP40 with brightness values corresponding to monomer - hexamer ( ~ 1 . - 1 . 6 ) , hexamer - 12mer ( ~ 1 . 6 - 2 . 0 ) , 12mer - 24mer ( 2 . 0 - 3 . 2 ) and > 24mer ( > 3 . 2 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 cell per biological replicate , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons compared to the control DMSO % average pixels ( * * p = 0 . 0035 ) . Data information : N & B : Number & Brightness analysis .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N&B analysis. D Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP-WT-eVP40 and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). E Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (~1.-1.6), hexamer-12mer (~1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9 cell per biological replicate, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons compared to the control DMSO % average pixels (**p=0.0035). Data information: N&B: Number & Brightness analysis.


A - D Functional budding assays assessed at 24 hours ( A - B ) and 48 hours ( C - D ) post treatment . A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by mouse α - GAPDH and HRP - Sheep α - Mouse ) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . C Representative western blot of budding assays performed at 48 hours post vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by ( Mouse α - GAPDH and HRP - Sheep α - Mouse ) D Quantification of relative budding index at 48 hours post fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . ( * p = 0 . 0260 ) Data information : VLPs : virus - like particles ; GAPDH : glyceraldehyde 3 - phosphate dehydrogenase ; HRP : horseradish peroxidase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Functional budding assays assessed at 24 hours (A-B) and 48 hours (C-D) post treatment. A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by mouse α-GAPDH and HRP-Sheep α-Mouse) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. C Representative western blot of budding assays performed at 48 hours post vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by (Mouse α-GAPDH and HRP-Sheep α-Mouse) D Quantification of relative budding index at 48 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. (*p=0.0260) Data information: VLPs: virus-like particles; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.


A - C TEM analysis of eVLP morphology . A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG - eVP40 and eGP following 48 hours of DMSO ( left panel ) or 5 µM fendiline treatment ( right panel ) . B Quantification of eVLP length ( µm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * * p = 0 . 0139 ) . C Quantification of eVLP diameter ( nm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * p = 0 . 0430 ) . Data information : TEM : transmission electron microscopy ; eVLPs : entry - competent VLPs ; eGP : Ebola virus

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

A-C TEM analysis of eVLP morphology. A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO (left panel) or 5 µM fendiline treatment (right panel). B Quantification of eVLP length (µm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). C Quantification of eVLP diameter (nm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430). Data information: TEM: transmission electron microscopy; eVLPs: entry-competent VLPs; eGP: Ebola virus glycoprotein


D , E Fluorescence based DiI TIM - 1 dependent entry assay . D Representative confocal images from the DiI - entry assay comparing entry of eVLPs produced from DMSO ( top panel ) and fendiline - treated HEK293 cells ( bottom panel ) into target cells ( HEK293 cells transiently expressing increasing amounts of TIM - 1 ; 0 . 0 µg , 0 . 5 µg , 1 . 0 µg ) . A stack of 10 frames was acquired for each image . DiI ( initially red ) was recolored to yellow for easier observation in print ; blue ( Hoechst 3342 stain ) ; scale bar = 10 µm . E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI - positive cells . Three images from each z - stack were quantified . N = 9 cell per replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons against the 0 . 0 µg TIM - 1 condition for both DMSO - and fendiline derived eVLPs . ( * * * * p < 0 . 0001 ; * * p = 0 . 0093 ) . Data information : TIM - 1 : t - cell immunoglobulin receptor - 1 ; eVLPs : entry - competent VLPs ; DiI : 1 , 1 ' - Dioctadecyl - 3 , 3 , 3 ' , 3 ' - Tetramethylindocarbocyanine Perchlorate .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E Fluorescence based DiI TIM-1 dependent entry assay. D Representative confocal images from the DiI-entry assay comparing entry of eVLPs produced from DMSO (top panel) and fendiline-treated HEK293 cells (bottom panel) into target cells (HEK293 cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI-positive cells. Three images from each z-stack were quantified. N=9 cell per replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1 condition for both DMSO- and fendiline derived eVLPs.(****p<0.0001; **p=0.0093). Data information: TIM-1: t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; DiI: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate.


B . Before differentiation , hESCs show uniformly high expression of OCT4 . Treatment with BMP4 produces a mixture of OCT4 + / CDX2 - self - renewing hESCs and OCT4 - / CDX2 + mesodermal cells .

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B. Before differentiation, hESCs show uniformly high expression of OCT4. Treatment with BMP4 produces a mixture of OCT4+/CDX2- self-renewing hESCs and OCT4-/CDX2+ mesodermal cells.


C . Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs . Cells were assigned to one of two distinct populations based on a 2 - component mixed Gaussian

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C. Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs. Cells were assigned to one of two distinct populations based on a 2-component mixed Gaussian distribution


E . Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration . Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity .

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E. Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity.


F . Distribution of OCT4 levels in individual hESCs . A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell .

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F. Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell.


G . Single - cell traces of OCT4 signaling . The length of each cell ' s trace indicates its cell cycle duration .

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G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration.


H . Distribution of cell cycle durations for 120 hESCs .

H. Distribution of cell cycle durations for 120 hESCs.


A . Single - cell traces of hESCs before and after treatment with 100 ng / mL BMP4 . Cells were imaged for 42 h prior to BMP4 treatment . Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division .

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A. Single-cell traces of hESCs before and after treatment with 100 ng/mL BMP4. Cells were imaged for 42 h prior to BMP4 treatment. Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division.


B . 24 h after BMP4 treatment , cells were fixed , stained for expression of CDX2 , and returned to the microscope for registration with the final time - lapse image . Mean nuclear OCT4 - mCherry and CDX2 were quantified for each cell , and the resulting distribution was fit to a 2 - component mixed Gaussian distribution representing self - renewing ( OCT4 + / CDX2 - ) and differentiated ( OCT4 - / CDX2 + ) cells hESCs that could be assigned to either distribution with > 99 % confidence ( red and green dots , Pdiff < 0 . 01 or Pdiff > 0 . 99 ) were considered for pro - fate analysis . Cells that did not reach this threshold ( gray dots ) were not used to determine pro - fate . Because cells in the final frame were assigned to only self - renewing or differentiated categories , pro - mixed

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B. 24 h after BMP4 treatment, cells were fixed, stained for expression of CDX2, and returned to the microscope for registration with the final time-lapse image. Mean nuclear OCT4-mCherry and CDX2 were quantified for each cell, and the resulting distribution was fit to a 2-component mixed Gaussian distribution representing self-renewing (OCT4+/CDX2-) and differentiated (OCT4-/CDX2+) cells hESCs that could be assigned to either distribution with >99% confidence (red and green dots, Pdiff < 0.01 or Pdiff > 0.99) were considered for pro-fate analysis. Cells that did not reach this threshold (gray dots) were not used to determine pro-fate. Because cells in the final frame were assigned to only self-renewing or differentiated categories, pro-mixed cells


C . Distributions of OCT4 levels in pro - self - renewing , pro - mixed , and pro - differentiated cell populations .

C. Distributions of OCT4 levels in pro-self-renewing, pro-mixed, and pro-differentiated cell populations.


D . Distributions of OCT4 pulse frequencies in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis .

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D. Distributions of OCT4 pulse frequencies in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.


E . Distributions of cell cycle durations in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis .

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E. Distributions of cell cycle durations in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.


F . Decision surfaces for logistic regression classifier . Cells with self - renewing ( red ) or differentiated ( green ) pro - fates are plotted above the predicted class surfaces . ( left ) Slice of decision surface for pulse frequency vs . OCT4 levels at a cell cycle duration of 14 h . ( right ) Slice of decision surface for cell cycle duration vs . OCT4 level at a pulse frequency of 0 . 25 h - 1 . * P < 0 . 05 , * * P < 0 . 0005 ; ns , not significant .

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F. Decision surfaces for logistic regression classifier. Cells with self-renewing (red) or differentiated (green) pro-fates are plotted above the predicted class surfaces. (left) Slice of decision surface for pulse frequency vs. OCT4 levels at a cell cycle duration of 14 h. (right) Slice of decision surface for cell cycle duration vs. OCT4 level at a pulse frequency of 0.25 h-1. * P < 0.05, ** P < 0.0005; ns, not significant.


A . Lineage of OCT4 expression dynamics . Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions . Vertical bars represent individual cells that completed an entire cell cycle lifetime . Thin horizontal bars denote cell division events . Color scale indicates low ( black ) , intermediate ( red ) , and high ( white ) OCT4 expression levels .

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A. Lineage of OCT4 expression dynamics. Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions. Vertical bars represent individual cells that completed an entire cell cycle lifetime. Thin horizontal bars denote cell division events. Color scale indicates low (black), intermediate (red), and high (white) OCT4 expression levels.


B . Differences in OCT4 levels between sister cells , cousin cells , second cousin cells , and randomly paired cells .

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B. Differences in OCT4 levels between sister cells, cousin cells, second cousin cells, and randomly paired cells.


C . Filmstrip showing distribution of OCT4 to daughter cells during cell division .

C. Filmstrip showing distribution of OCT4 to daughter cells during cell division.


D . Distribution of OCT4 ratios between sister cells before and after BMP4 treatment . Ratios for both sister cells ( r and 1 / r ) are plotted to emphasize central tendency toward r = 1 : 1 . Differently colored curves represent the distribution of ratios at different time points after division . For time points after 5 minutes , the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters .

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D. Distribution of OCT4 ratios between sister cells before and after BMP4 treatment. Ratios for both sister cells (r and 1/r) are plotted to emphasize central tendency toward r = 1:1. Differently colored curves represent the distribution of ratios at different time points after division. For time points after 5 minutes, the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters.


E . Proliferating H9 - OCT4 - mCherry hESCs were imaged for 24 h , fixed , and stained for DNA content with DAPI . The probability density of DAPI ratio ( blue ) and OCT4 - mCherry ratio ( red ) between sister cells that divided ≤ 1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function . A two - sample Kolmogorov - Smirnov test was used to determine significance . * P < 0 . 05 , * * * P < 0 . 0005 , two - sample Kolmogorov - Smirnov test ; ns , not significant .

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E. Proliferating H9-OCT4-mCherry hESCs were imaged for 24 h, fixed, and stained for DNA content with DAPI. The probability density of DAPI ratio (blue) and OCT4-mCherry ratio (red) between sister cells that divided ≤1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function. A two-sample Kolmogorov-Smirnov test was used to determine significance. * P < 0.05, *** P < 0.0005, two-sample Kolmogorov-Smirnov test; ns, not significant.


B . OCT4 mixing as a function of cell history . 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division .

B. OCT4 mixing as a function of cell history. 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division.


C . Variance in final OCT4 expression explained over time during maternal , inherited , and autonomous periods of cell history . At each time point , the coefficient of determination ( R2 ) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated .

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C. Variance in final OCT4 expression explained over time during maternal, inherited, and autonomous periods of cell history. At each time point, the coefficient of determination (R2) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated.


D . Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells . The same R2 over time mother - daughter traces were randomly sampled from a set of mother - daughter traces that contained sister - cell pairs ( blue line ) or only one randomly selected sister cell from each pair ( red line ) . E . Difference in variance explained between red and blue traces in panel D . The black line represents the actual difference ; the red line shows a smoothing ( moving average = 1 . 25 h ) of the black trace to highlight the overall trend .

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D. Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells. The same R2 over time mother-daughter traces were randomly sampled from a set of mother-daughter traces that contained sister-cell pairs (blue line) or only one randomly selected sister cell from each pair (red line). E. Difference in variance explained between red and blue traces in panel D. The black line represents the actual difference; the red line shows a smoothing (moving average = 1.25 h) of the black trace to highlight the overall trend.


Meth Induces Rapid Alkalization of Acidic Organelles , and Changes in Endosomal Structure in Dendritic Cells ( A ) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment . Similar results were obtained in three independent experiments . ( Statistics n = 25 - 35 , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . )

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Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)


( B ) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence ( ) or presence ( ▴ ) of 50 μM Meth . p < 0 . 001 for all time points versus control ( n = 25 - 35 , mean ± SEM , Student ' s two - tailed t - Test ) .

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(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test).


( C ) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement . ( Statistics n = 30 - 90 cells , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . )

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(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)


( D ) Representative electron micrographs showing multivesicular endosomal compartments ( arrows ) in control , Clq - and Meth - treated cells at 4 h . Scale bar , 200 nm .

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(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm.


( E ) Ultra - thin cryosections of control , Clq - and Meth - treated dendritic cells as in ( D ) , immunolabeled for LAMP - 1 ( 15 nm gold particles ) and MHC class II ( 10 nm gold particles ) . Scale bar , 200 nm .

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(E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.


( A ) Accumulation of GFP - LC3 labeled autophagosomes in Meth - or Clq - treated dendritic cells at 2 h and 24 h . Fisher ' s exact test P values versus control ( n = 300 - 400 cells , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 0001 ) .

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(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).


( B ) GFP - LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment . Similar results were obtained in three independent experiments . Fisher ' s exact test versus control ( n = 300 - 400 cells , mean ± SEM , * * * p < 0 . 0001 ) .

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(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001).


( C ) Representative fluorescence micrographs of GFP - LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h . Arrow denotes examples of autophagosomes . Scale bar , 10 μm .

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(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.


( A ) Cells were incubated in indicated concentrations of Meth or Clq for 2 h , and then IgG coated erythrocytes were added . Extracellular uningested EIgG were lysed and removed . Phagocytic index ( PI ) was quantified and inhibition is indicated as percent of control . Data were collected from four to seven experiments ( n = 300 cells , mean ± SEM , * * * p < 0 . 0001 , two - tailed ANOVA ) .

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(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).


( B ) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment . Arrows denote phagocytosed erythrocytes . Scale bar , 10 μm .

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(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.


( C , D ) J774 . 16 cells were exposed to PBS , chloroquine , or methamphetamine for 2 h followed by incubation with Cn or Ca . The phagocytic indices ( ratio of intracellular yeast to the number of macrophages counted ) were determined after 1 h or 30 min for Cn and Ca , respectively ( C ) . ( Statistics n = 300 , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) CFU determinations were preformed after 24 h incubations ( D ) . The values are the mean and standard deviation from determinations made in triplicate wells ( n = 300 cells , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) . Experiments were repeated three times with similar results .

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(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.


( A ) After randomized , 8 - 12 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 30 mg / kg ) for 12 hours via intraperitoneal injection . Serum was collected and ELISA assays were used to detect the level of IL - 1β ( n = 6 , 10 , 6 , and 12 mice for each group , respectively ) . Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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(A) After randomized, 8-12 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (30 mg/kg) for 12 hours via intraperitoneal injection. Serum was collected and ELISA assays were used to detect the level of IL-1β (n = 6, 10, 6, and 12 mice for each group, respectively). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p< 0.0001 (Student's t-test).


( B ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated by 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed and the culture medium was collected . Western blotting ( WB ) assays were used to detect protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) .

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(B) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated by 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed and the culture medium was collected. Western blotting (WB) assays were used to detect protein levels in whole cell lysates (Lys) and culture medium (Sup).


( C ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated with 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . ELISA assays were used to detect protein levels in the culture medium . Data of BMDMs from the separate mice ( n = 6 mice for each group ) and are presented as mean ± SEM . * * * p < 0 . 001 ( Student ' s t - test ) .

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(C) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated with 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. ELISA assays were used to detect protein levels in the culture medium. Data of BMDMs from the separate mice (n = 6 mice for each group) and are presented as mean ± SEM. *** p < 0.001 (Student's t-test).


( D ) After randomized , 4 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 300 mg / kg ) to induce septic shock via intraperitoneal injection . The life span of Phb1F / F mice ( n = 6 ) and Phb1MyeKO mice ( n = 7 ) was recorded .

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(D) After randomized, 4 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (300 mg/kg) to induce septic shock via intraperitoneal injection. The life span of Phb1F/F mice (n = 6) and Phb1MyeKO mice (n = 7) was recorded.


( E ) PBMCs were extracted from normal controls and patients with sepsis ( n = 6 individuals ) . Intracellular mRNA levels were detected by real - time quantitative PCR ( RT - qPCR ) assays ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) .

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(E) PBMCs were extracted from normal controls and patients with sepsis (n = 6 individuals). Intracellular mRNA levels were detected by real-time quantitative PCR (RT-qPCR) assays (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01 (Student's t-test).


( F ) PBMCs were extracted from normal controls and infant patients with sepsis . Protein levels in whole cell lysates were detected by WB assays .

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(F) PBMCs were extracted from normal controls and infant patients with sepsis. Protein levels in whole cell lysates were detected by WB assays.


J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours Intracellular mRNA levels were detected by RT - qPCR assays . Data are from four independent experiments for ( G ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours Intracellular mRNA levels were detected by RT-qPCR assays. Data are from four independent experiments for (G) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).


J774A . 1 cells were stimulated with recombinant IL - 1β ( 100 pg / ml ) for 24 hours . Intracellular mRNA levels were detected by RT - qPCR assays . Data are from six independent experiments for ( H ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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J774A.1 cells were stimulated with recombinant IL-1β (100 pg/ml) for 24 hours. Intracellular mRNA levels were detected by RT-qPCR assays. Data are from six independent experiments for (H) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).


( I ) After J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 0 , 3 , 6 , 12 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays .

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(I) After J774A.1 cells were stimulated with LPS (200 ng/ml) for 0, 3, 6, 12 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.


( J ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays .

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(J) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.


( K ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the culture medium was collected . ELISA assays were used to detect IL - 1β levels in the culture medium . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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(K) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the culture medium was collected. ELISA assays were used to detect IL-1β levels in the culture medium. Data are from three independent experiments (mean ± SEM). n.s., no significance, **** p < 0.0001 (Student's t-test).


( A - B ) GFP - expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence ( IF ) analysis to detect mitochondria ( MitoTracker , red ; A ) or ΔΨm ( TMRM , red ; B ) . Images shown are representative of at least three independent experiments . White boxed regions in the panels in ( A ) are enlarged . Scale bar : 10 μm .

(A-B) GFP-expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.


( C ) Flow cytometric analysis of ΔΨm ( TMRM ) . HeLa cells with stable Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes or FCCP ( 4 μM ) for 5 minutes .

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(C) Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 minutes or FCCP (4 μM) for 5 minutes.


( D ) The mitochondrial respiratory capacity of J774A . 1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer . Data are from at least three independent experiments ( mean ± SEM ) . n . s . , no significance . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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(D) The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).


( E ) HeLa cells with stable PHB1 ablation were incubated with H2O2 ( 2 μM ) and CsA ( 2 μM ) for 30 minutes . Flow cytometric analysis of intracellular ROS levels ( DHE ) .

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(E) HeLa cells with stable PHB1 ablation were incubated with H2O2 (2 μM) and CsA (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS levels (DHE).


( F - G ) Confocal microscopy analyses of mitochondrial Ca2 + in time - series mode . HeLa cells stably expressing 4mt - RCaMPh with or without Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes . During acquisition of fluorescence images ( F ) , cells were treated with histamine ( 200 μM ) . Traces of mitochondrial Ca2 + ( 4mt - RCaMPh ) dynamics are shown in ( G ) ( mean ± SEM ) . n = 16 , 16 , 15 , 16 ( from the top to the bottom ) . Scale bar : 10 μm . * p < 0 . 05 relative to Scramble group , # p < 0 . 05 relative to shPHB1 group ( Student ' s t - test ) .

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(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa cells stably expressing 4mt-RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).


( A ) The co - localization between mtDNA ( SG - ALK , green ) and mitochondria ( MitoTracker , red ) was examined in J774A . 1 cells with or without knockdown of Phb1 or Phb2 . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative analysis of data from ( A ) . Data are from ten , eight , and seven images from three independent experiments ( left to right , respectively ) ( mean ± SEM ) . n . s . , no significance , * * * p < 0 . 001 ( Student ' s t - test ) .

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(A) The co-localization between mtDNA (SG-ALK, green) and mitochondria (MitoTracker, red) was examined in J774A.1 cells with or without knockdown of Phb1 or Phb2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from ten, eight, and seven images from three independent experiments (left to right, respectively) (mean ± SEM). n.s., no significance, *** p < 0.001 (Student's t-test).