( D ) Representative images of COS7 cells showing that GFP - COP1 ( green ) and TRIB1 - HA ( red ) are diffuse in the nucleoplasm when coexpressed with C / EBPα ( cyan ) . Cells were stained with anti - HA ( Santa Cruz ) , anti - C / EBPα ( Cell Signaling ) , anti - mouse Alexa Fluor 405 , and anti - rabbit Alexa Fluor 680 . Scale bar , 10 µm . See also Fig EV2C for images of cells expressing each construct alone or in pairs .

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

(D) Representative images of COS7 cells showing that GFP-COP1 (green) and TRIB1-HA (red) are diffuse in the nucleoplasm when coexpressed with C/EBPα (cyan). Cells were stained with anti-HA (Santa Cruz), anti-C/EBPα (Cell Signaling), anti-mouse Alexa Fluor 405, and anti-rabbit Alexa Fluor 680. Scale bar, 10 µm. See also Fig EV2C for images of cells expressing each construct alone or in pairs.


( A ) Representative images of COS7 cells expressing COP1 - 3xFLAG ( green ) co - transfected with empty vector , GFP , or the indicated GFP - TRIB1 constructs ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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 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

(A) Representative images of COS7 cells expressing COP1-3xFLAG (green) co-transfected with empty vector, GFP, or the indicated GFP-TRIB1 constructs (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of COP1 - 3xFLAG in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of COP1-3xFLAG in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (*p < 0.01; **p < 0.001; ***p < 0.0001).


( C ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector , TRIB1 - FLAG , TRIB2 - FLAG , or TRIB3 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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

(C) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector, TRIB1-FLAG, TRIB2-FLAG, or TRIB3-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( ns , not significant ; * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (ns, not significant; ***p < 0.0001; ****p < 0.00001).


( B ) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP - COP1 constructs and untagged TRIB1 .

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

(B) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP-COP1 constructs and untagged TRIB1.


( C ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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

(C) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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 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

(D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (***p < 0.0001; ****p < 0.00001).


( B ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 4A ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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

(B) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 4A (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * * p < 0 . 00001 ) .

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 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

(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (****p < 0.00001).


( D ) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL , COP1 PSL 4A , and TRIB1 tail peptides to compete with FITC - TRIB1 tail for binding to the COP1 WD40 domain ( 376 - 731 ) . The COP1 WD40 domain was preincubated with FITC - TRIB1 tail before addition of the indicated concentrations of unlabeled peptide . The change in polarization is plotted as a function of unlabeled peptide concentration . Curves were fit to a sigmoidal dose - response curve . Data are presented as mean values ± S . E . M . for three independent experiments where each measurement was made in duplicate . .

O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL, COP1 PSL 4A, and TRIB1 tail peptides to compete with FITC-TRIB1 tail for binding to the COP1 WD40 domain (376-731). The COP1 WD40 domain was preincubated with FITC-TRIB1 tail before addition of the indicated concentrations of unlabeled peptide. The change in polarization is plotted as a function of unlabeled peptide concentration. Curves were fit to a sigmoidal dose-response curve. Data are presented as mean values ± S.E.M. for three independent experiments where each measurement was made in duplicate..


( F ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and empty vector or TRIB1 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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 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

(F) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and empty vector or TRIB1-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( G ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( F ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant , * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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

(G) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (F). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant, ***p < 0.0001; ****p < 0.00001).


( A ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 NESmut ( V238A / L242A ) ( green ) and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) and anti - rabbit Alexa Fluor 680 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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

(A) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 NESmut (V238A/L242A) (green) and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling) and anti-rabbit Alexa Fluor 680. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( B ) Representative images of COS7 cells transfected with GFP - COP1 WT ( green ) , the indicated TRIB1 - HA constructs ( red ) , and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) , anti - HA ( Sigma ) , anti - rabbit Alexa Fluor 680 , and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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 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

(B) Representative images of COS7 cells transfected with GFP-COP1 WT (green), the indicated TRIB1-HA constructs (red), and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling), anti-HA (Sigma), anti-rabbit Alexa Fluor 680, and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( A ) and ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * p < 0 . 01 ; * * * * p < 0 . 00001 ) .

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

(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (A) and (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; *p < 0.01; ****p < 0.00001).


( E ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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

(E) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( F ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( E ) and Figure EV6C . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * * * * p < 0 . 00001 ) .

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

(F) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (E) and Figure EV6C. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; ****p < 0.00001).


( G ) Quantification of percentage of cells observed in ( E ) and Figure EV6C with the indicated GFP - COP1 construct exhibiting nuclear ( Nuc ) , nuclear and cytoplasmic ( Nuc / Cyt ) , or cytoplasmic ( Cyt ) localization . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed .

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

(G) Quantification of percentage of cells observed in (E) and Figure EV6C with the indicated GFP-COP1 construct exhibiting nuclear (Nuc), nuclear and cytoplasmic (Nuc/Cyt), or cytoplasmic (Cyt) localization. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed.


B . Western blot of oocyte lysate ( left ) and biotinylation - purified surface ( right ) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1 / PC2 complex compared to either protein expressed separately . Anti - PC1 C - terminus antibody [ 29 ] recognized both full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 . A higher - glycosylated 130 kDa PC2 ( star ) band was only seen when PC1 is coexpressed .

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

B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.


C . Co - IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes . IP was done with an anti - FLAG antibody . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 are indicated . Both 120 and 130 kDa bands of PC2 were seen in the IPed product .

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

C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.


D . Representative current - voltage relationship ( I - V ) curves ( left ) and a scatter plot and bar graph ( right ) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording . The current of the GOF PC2 _ F604P is included as a control . Currents at + 60 mV are shown in the bar graph . Each point represents the recorded current from one oocyte . Oocyte numbers for scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O 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 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

D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).


B . Representative I - V curves ( left ) and a scatter plot and bar graph ( right ) showing that the PC2 _ AA is a GOF mutant and gave rise to a larger current than PC2 _ F604P . Scatter plot and bar graph shows the average current sizes at + 60 mV . The cations included in the bath solution , 100 mM Na + and 2 mM Ca2 + in this case , are indicated by the thick - lined boxes here and in all the following figures . Oocyte numbers for bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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 O

B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


C . Representative currents of PC2 _ F604P and PC2 _ AA mutants in the divalent ion - free bath solution , which contains 100 mM Na + .

O O O O O B-CONTROLLED_VAR 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

C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.


D . Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2 + .

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

D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.


E . Calcium - activated chloride channel ( CaCC ) blocker MONNA ( 10 μM ) or niflumic acid ( NFA ) ( 1 mM ) partially blocked the current recorded from the PC2 _ AA - injected oocytes in the 70 mM Ca2 + bath solution .

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

E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.


A , B . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 100 mM Na + ( A ) , and 100 mM Na + and 2 mM Ca2 + ( B ) . Scatter plot and bar graphs show currents at + 80 mV and - 80 ( or - 70 ) mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) .

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

A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).


C . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 100 mM Na + and 2 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

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

C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


D . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 70 mM Ca2 + . Scatter plot and bar graphs show currents at + 80 mV and - 70 mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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

D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


E . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 70 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

O O O O O O O O O O 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. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


F . Representative currents of PC2 _ AA ( left ) and PC1 / PC2 _ AA ( right ) in solutions containing the indicated Ca2 + concentrations . All solutions contain 20 μM of MONNA to block CaCC current . Corresponding concentrations of NMDG + were added to compensate the osmolarity . See Fig EV2 for currents of the full voltage scale and the details of solutions used .

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

F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.


G . Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1 / PC2 _ AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone . For oocytes injected with PC2 alone , five times more concentrated PC2 RNA was injected to increase its surface expression . The purple dashed line indicates the background 45Ca uptake set by the measurement with the water - injected oocytes . Data were averaged from three independent experiments . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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

G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


H . Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments .

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

H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + ( B ) , Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

O O O 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 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

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( C ) Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

O O O 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 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

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 70 mM Ca2 + ( D ) . Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


E . Scatter plot and bar graph showing that the current of PC1 - CTF / PC2 _ AA is completely abolished when WT PC2 was used , or after introducing R4090W mutation in the putative pore region of PC1 - CTF . Inserted are western blot images showing the expression of the corresponding proteins . Top : anti - PC1 . Bottom : anti - PC2 . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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

E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + and 2 mM Ca2 + ( F ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + ( G ) , , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2 + ( H ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


I . Co - IP followed by western blot showing the association between PC2 _ AA and the indicated full - length or fragments of PC1 . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

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 O O O O 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

I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


J . Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2 _ AA and full - length PC1 , PC1 - CTF , or PC1 - TLD at the oocyte surface . Besides the blots shown in the figure , surface samples were also blotted with an anti - PC1 N - terminus antibody , and the cleaved NTF fragment was found associated to the plasma membrane in the full - length sample ( shown in Appendix Fig S4 ) . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

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

J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


A . Representative I - V curves of the PC2 _ AA and PC1 - CTF / PC2 _ AA channels in divalent ion - free bath solutions containing 100 mM of indicated ions , showing the differences in reversal potential .

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

A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.


B . Bar graphs showing the markedly different reversal potentials of PC2 _ AA channel and the PC1 - CTF / PC2 _ AA channel in bath solutions containing 100 mM of the indicated ions . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) .

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

B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( C ) These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

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

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( D ) . These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

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

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions ( B ) , indicating that these amino acids are essential for ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

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

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others ( C ) , indicating a less important role of these amino acids in ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

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

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( D ) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

O O O 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 O O O O O O O O

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( E ) . Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

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

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( B ) Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

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 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 B-CONTROLLED_VAR 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

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 70 mM Ca2 + ( C ) . Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

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 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 B-CONTROLLED_VAR 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

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


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

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

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 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 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

(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 B-MEASURED_VAR 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 O O O O O O O O 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 .

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

(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 .

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

(D) The 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 .

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

(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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O 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-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 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

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 .

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

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 .

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

(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 .

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

(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 .

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) 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 .

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

(C) (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 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 O O O O O O O O O O O O O O O O O O O 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 .

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

(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 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

(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 B-CONTROLLED_VAR 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 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 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 B-CONTROLLED_VAR 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 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 B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 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 .

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

O O O O B-CONTROLLED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) Silencing ATF4 markedly decreased the migration of BGC823 cells with UHMK1 overexpression. (left panel) The quantitative number of migrated cells in scramble+Flag, scramble+Flag-UHMK1, siATF4+Flag and siATF4+Flag-UHMK1 group. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (right panel) Representative images of migrated cells in each group are shown. Scale bars=100μm. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


( G ) Silencing ATF4 markedly decreased the invasion of UHMK1 overexpressing BGC823 cells . ( left panel ) The quantitative number of invaded cells in scramble + Flag , scramble + Flag - UHMK1 , siATF4 + Flag and siATF4 + Flag - UHMK1 group . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - tailed statistical significance was assessed by Student ' s t ‐ test . ( right panel ) Representative images of invaded cells in each group are shown . Scale bars = 100μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O B-CONTROLLED_VAR 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 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

(G) Silencing ATF4 markedly decreased the invasion of UHMK1 overexpressing BGC823 cells. (left panel) The quantitative number of invaded cells in scramble+Flag, scramble+Flag-UHMK1, siATF4+Flag and siATF4+Flag-UHMK1 group. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (right panel) Representative images of invaded cells in each group are shown. Scale bars=100μm. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


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

O O O O O 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 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

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


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

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


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

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

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


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

O O O O O 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

(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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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 .

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

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


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

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

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


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

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

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

(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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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

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

(B) Overexpression of ATF4 enhanced the expression of UHMK1 in BGC823 and HGC27 cells. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.


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

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

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


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

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

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


( F ) ATF4 deficiency and overexpression regulated the activity of the UHMK1 promoter in GC cells . Data information : Data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 .

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

(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 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

(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

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

(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 , .

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

(C) 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 .

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

(C) (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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