( A - C ) Distribution of spindle pole factors in centrosomal and acentrosomal spindles . DMSO - treated control mitotic spindles ( 2 - centrosomes ) and centrinone - treated acentrosomal spindles ( 0 - centrosome ) of HeLa cells . ( A ) Red , gray , green , and blue represent NuMA , α - tubulin , Cep152 , and DNA , respectively . Z - projections of eight sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Red , gray , and blue represent NuMA , Katanin p60 , and DNA , respectively . Z - projections of five sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Red , gray , and blue represent p150glued , ASPM , and DNA , respectively . Z - projections of 10 ( 2 - centrosomes ) or five ( 0 - centrosome ) sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.


( D ) The structure of NuMA and microtubules in centrinone - treated acentrosomal spindles of HeLa cells . Two - way arrows indicate the bipolarity of elongated NuMA structures . Gray , red , green and blue represent α - tubulin , NuMA , Cep152 and DNA , respectively . Scale bar , 5 μm .

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

(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.


( E ) The localization of p150glued in centrinone - treated acentrosomal spindles of HeLa cells . Red , gray , green and blue represent NuMA , p150glued , Cep152 and DNA , respectively . Scale bar , 5 μm .

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

(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.


( F ) NuMA distribution in 3D . Images of NuMA ( Red ) and chromosomes ( blue ) were reconstructed in 3D and surface - rendered .

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 O O O O

(F) NuMA distribution in 3D. Images of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered.


( G ) Representative images of NuMA ( arrowheads ) in centrosomal and acentrosomal cells before and after photobleaching . DMSO - or centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed . Time after photobleaching ( sec ) is indicated . Scale bar , 5 μm .

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

(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.


( H ) Normalized fluorescence intensities of NuMA - mClover at centrosomal poles ( control , 2 - centrosomes ) or at acentrosomal spindle pole ( 0 - centrosome ) during the FRAP time course ( N = 20 cells for each condition ) . The first three time points are before photobleaching . Error bars show standard deviation ( SD ) .

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

(H) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD).


( I ) Mobile fractions from the normalized recovery curves of NuMA - mClover at centrosomal spindle poles ( control , 2 - centrosomes ) or at acentrosomal spindle poles ( 0 - centrosome ) . Mobile fractions ( roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting . Line and error bars represent the median and interquartile range ( N = 20 cells for each condition ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

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

(I) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


( A ) Time - lapse observation of the establishment of bipolarity in acentrosomal cells . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads indicate the assembling NuMA after NEBD . Two - way arrows indicate the bipolarity . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

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

(A) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( B ) Time - lapse observation of the dynamics of NuMA and chromosomes . Centrinone - treated HeLa cells expressing mCover - NuMA were observed with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads and two - way arrows indicate the assembling NuMA after NEBD and bipolarity , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

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 B-MEASURED_VAR 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

(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( C ) The timing of bipolarity establishment in the acentrosomal pole , chromosome alignment , and anaphase onset Each plot shows the cumulative percentage of each event at individual time points ( N = 49 cells from two independent 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 O O O O O O O O O O O O O O O

(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).


( D ) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells . Two - way arrow indicates the bipolarity of elongated NuMA structures . Red , gray , green , and blue represent NuMA , CENP - C , GT335 , and DNA , respectively . Scale bar , 5 μm .

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

(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.


( E , F ) Time - lapse observation of the structure of NuMA and microtubules upon centrosome removal . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed with a 60 × objective . Red and green represent NuMA and SiR - tubulin , respectively . Z - projections of 17 sections , every 1 μm . Scale bar , 5 μm . Time zero corresponds to NEBD . ( F ) An example of NuMA formed several asters ( arrowheads ) at the time of NEBD .

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

(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.


( A ) NuMA structure upon microtubule depolymerization . Nocodazole - treated HCT116 CMV - OsTIR1 CEP152 - mClover - mAID cells were treated with 100 ng / ml nocodazole . Gray , red , green and blue represent α - tubulin , NuMA , CEP152 ( mClover ) and DNA , respectively . Scale bar , 5 μm .

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

(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.


( B ) Immunostaining of DHC1 and NuMA in centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells . Gray , green , red and blue represent DHC1 ( mClover ) , GT335 , NuMA , and DNA , respectively . Scale bar , 5 μm .

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

(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.


( D ) NuMA structure upon DHC1 depletion . Centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM indole - 3 - acetic acid ( IAA ) . Arrowhead indicates the spindle pole . Green , red , gray and blue represent DHC1 ( mClover ) , NuMA , GT335 and DNA , respectively . Scale bar , 5 μm . ( E ) Frequency of acentrosomal pole patterns upon DHC1 depletion in ( D ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 24 spindles in each experiment ) . (

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

(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). (


( F ) The distribution of NuMA upon DHC1 depletion . Arrowheads indicate the presence of NuMA on microtubules . Green , red , and blue represent α - tubulin , NuMA , and DNA , respectively . Scale bars , 5 μm .

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(F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.


( G ) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA - mAID - mClover - FLAG with either mCherry - NuMA WT or 5A - 3 . Arrowheads indicate the assembled NuMA structure . Green , red , gray and blue represent endogenous NuMA ( mClover ) , expressed NuMA ( mCherry ) , GT335 and DNA , respectively . Z - projections of 21 sections , every 1 μm . Scale bar , 5 μm . ( H ) The number of NuMA structure in ( G ) . Values are mean percentages ± SD . from four independent experiments ( N ≧ 15 spindles in each experiment ) . One - way ANOVA with Tukey ' s multiple comparisons test was used to obtain a P value .

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 B-MEASURED_VAR 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 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

(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.


( A , B ) Structure of the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM IAA . Two - way arrow indicates the bipolarity . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Frequency of spindle structure patterns upon NuMA depletion in ( A , B ) . Values are mean percentages ± SD . from three independent experiments ( N = 30 spindles in each experiment ) . (

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 B-MEASURED_VAR I-MEASURED_VAR I-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 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

(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment). (


( D ) The localization of Eg5 in centrinone - treated acentrosomal HeLa cells . Red , gray , green and blue represent NuMA , Eg5 , GT335 and DNA , respectively . Scale bar , 5 μm .

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(D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.


( E ) The localization of Eg5 in the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with Dox and 500 μM IAA . Gray , green , red , and blue represent Eg5 , NuMA ( mClover ) , CP110 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

O O O O O O B-MEASURED_VAR 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 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 O

(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.


( F ) Structure of the spindle upon Eg5 inhibition . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 30 μM monastrol . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( G ) Frequency of spindle structure patterns upon Eg5 inhibition in ( F ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 23 spindles in each experiment ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-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 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

(F) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).


( A ) Time - lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5 . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed in the presence of 50 μM of monastrol with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads and two - way arrows indicate the monopolar and bipolar states , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( A ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 75 cells from two independent experiments ) . (

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 B-MEASURED_VAR 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

(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments). (


( C ) Time - lapse observation of the dynamics of mClover - NuMA and chromosomes . HeLa cells expressing mClover - NuMA were observed in the presence of SiR - DNA with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads indicate initial bipolarity in the acentrosomal spindle pole ( 8 min ) , dispersed chromosomes ( 328 min ) , and the monopolar - like state of the acentrosomal spindle pole ( 332 min ) , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( D ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( C ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 35 cells from three independent experiments ) .

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

(C) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).


( A ) Structure of the spindle upon NuMA and Eg5 inhibition . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline ( Dox ) , 500 μM IAA and monastrol . Green , red , and blue represent NuMA ( mClover ) , α - tubulin , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in ( A ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 22 spindles in each experiment ) . (

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(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment). (


( C ) The localization of Eg5 in the spindle upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline Dox and 500 μM IAA . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , Eg5 and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

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(C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.


( D ) Time - lapse observation of the establishment of spindle bipolarity upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml Dox and 500 μM IAA and observed with a 40 × objective . Red and green represent SiR - tubulin and NuMA , respectively . Z - projections of 14 sections , every 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( E ) The time required for the initial establishment of spindle bipolarity in ( D ) . Line and error bars represent the mean and SD . ( N ≧ 60 cells from two independent experiments ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

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(D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


A . Expression profiling of differentially expressed circRNAs between the tumor and normal groups . Rows represent circRNAs , and columns represent samples . Rows were ordered by fold change , and columns were ordered by their group . The sample of N8 was not included due to low sequencing library size .

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A. Expression profiling of differentially expressed circRNAs between the tumor and normal groups. Rows represent circRNAs, and columns represent samples. Rows were ordered by fold change, and columns were ordered by their group. The sample of N8 was not included due to low sequencing library size.


B . Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups . Both the row and column were unsupervised and clustered with the hierarchical clustering method .

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B. Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups. Both the row and column were unsupervised and clustered with the hierarchical clustering method.


C . The 4 of 22 differentially expressed circRNAs were confirmed by qRT - PCR , which were retained after marker selection procedure . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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C. The 4 of 22 differentially expressed circRNAs were confirmed by qRT-PCR, which were retained after marker selection procedure. **P < 0.01, Student's t-test, mean ± SD.


E . Time - dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset . P - values are shown for the indicated comparison of AUC between each marker and cirScore . Student ' s t - test , AUC = area under the curve .

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E. Time-dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset. P-values are shown for the indicated comparison of AUC between each marker and cirScore. Student's t-test, AUC=area under the curve.


A - F . Kaplan - Meier curves of DFS ( A ) and OS ( B ) in 249 patients in the training set . Kaplan - Meier curves of DFS ( C ) and OS ( D ) in 122 patients in the internal validation data set . Kaplan - Meier curves of DFS ( E ) and OS ( F ) in 180 patients in the external independent validation data set . Hazard ratios ( HRs ) were calculated with a univariate Cox regression analysis , P - values were calculated with the log - rank test .

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A-F. Kaplan-Meier curves of DFS (A) and OS (B) in 249 patients in the training set. Kaplan-Meier curves of DFS (C) and OS (D) in 122 patients in the internal validation data set. Kaplan-Meier curves of DFS (E) and OS (F) in 180 patients in the external independent validation data set. Hazard ratios (HRs) were calculated with a univariate Cox regression analysis, P-values were calculated with the log-rank test.


A . RT - PCR products with divergent and convergent primers showing circularization of has _ circ _ 0079480 and has _ circ _ 0087391 . cDNA , complementary DNA ; gDNA , genomic DNA .

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A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA.


B . qRT - PCR evaluated the knockdown efficiency of has _ circ _ 0079480 and and has _ circ _ 0087319 in SW620 and HCT116 cells transfected with two unique shRNAs ( # 1 , # 2 ) . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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B. qRT-PCR evaluated the knockdown efficiency of has_circ_0079480 and and has_circ_0087319 in SW620 and HCT116 cells transfected with two unique shRNAs (#1, #2). **P < 0.01, Student's t-test, mean ± SD.


C . Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs .

C. Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs.


D . The relative fold - change of the transwell migration for indicated knockdown cells over those of control cells . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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D. The relative fold-change of the transwell migration for indicated knockdown cells over those of control cells. **P < 0.01, Student's t-test, mean ± SD.


E - F . Representative hematoxylin and eosin ( H & E ) staining and statistical results of the micro - metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


G - H . Representative H & E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days . ( Five sections evaluated per lung ) . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


B . Still images of GFP ( syncytia ) and Propidium Iodide ( PI ) ( cell death ) at different time - points . Scale bar : 100 µm .

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B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm.


C . Quantification of U2OS - ACE2 fusion and death by time - lapse microscopy . Results are mean ± sd from 3 fields per condition .

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C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean±sd from 3 fields per condition.


D . S staining of infected U2OS - ACE2 cells analyzed by immunofluorescence . The Hoechst dye stains the nuclei . Scale bar : 40 µm .

D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm.


E . Surface S staining of infected U2OS - ACE2 cells analyzed by flow cytometry . Results are representative of at least three independent experiments .

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E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments.


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . A . TMPRSS2 increases fusion and cell mortality . Right panel : Areas of GFP + cells and PI +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . B . IFITM1 , but not IFITM2 and 3 , inhibits SARS - CoV - 2 - induced syncytia formation . Right panels : Area of GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . C . The fusion of IFITM1 + cells with U2OS - ACE2 syncytia is drastically reduced . Right panel : Area of IFITM + GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . D . IFITM1 decreases the number of infected cells . Right panel : Fraction of cells positive for S , normalized to control cells ( transduced with pQCXIP - empty vector ) .

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector ).


B . Representative images of cell - cell fusion . Scale bar : 100 µm .

B. Representative images of cell-cell fusion. Scale bar: 100 µm.


C . Quantification of GFP + areas . Results are mean ± sd from 5 independent experiments .

C. Quantification of GFP+ areas. Results are mean±sd from 5 independent experiments.


D . TMPRSS2 accelerates fusion . Cells were monitored by video microscopy and the GFP area was quantified over time . Left panel : one representative experiment . Results are mean ± sd from 3 fields per condition . Right panel : Mean ± sd from 7 independent experiments ( at 12h post transfection ) .

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D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean±sd from 3 fields per condition. Right panel: Mean±sd from 7 independent experiments (at 12h post transfection).


E . Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S - expressing 293T cells . One representative out of three independent experiment is shown .

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E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown.


A . S - expressing cells ( Donor cells ) , ACE2 - expressing cells ( Acceptor cells ) were co - transfected with TMPRSS2 , IFITM or control plasmids . Cell fusion was quantified by measuring the GFP + area after 18h . The indicated combinations were tested . The impact of IFITMs was measured in absence of TMPRSS2 ( left panel ) , in presence of TMPRSS2 in donor ( middle panel ) or acceptor cells ( right panel ) . Data are mean ± sd of 4 independent experiments . Statistical analysis : One - Way ANOVA , ns : non - significant , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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A. S-expressing cells (Donor cells), ACE2-expressing cells (Acceptor cells) were co-transfected with TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area after 18h. The indicated combinations were tested. The impact of IFITMs was measured in absence of TMPRSS2 (left panel), in presence of TMPRSS2 in donor (middle panel) or acceptor cells (right panel). Data are mean±sd of 4 independent experiments. Statistical analysis: One-Way ANOVA, ns: non-significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.


Impact of TMPRSS2 on IFITMs levels . B . TMPRSS2 does not decrease IFITM amounts measured by flow cytometry . 293T cells were transfected with the indicated IFITM plasmids , with or without TMPRSS2 , and analyzed 18h later .

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Impact of TMPRSS2 on IFITMs levels. B. TMPRSS2 does not decrease IFITM amounts measured by flow cytometry. 293T cells were transfected with the indicated IFITM plasmids, with or without TMPRSS2, and analyzed 18h later.


C . Impact of TMPRSS2 on S and ACE2 , measured by Western Blot . 293T cells were transfected with or without IFITM1 , TMPRSS2 , S or ACE2 plasmids , and analyzed 18h later .

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C. Impact of TMPRSS2 on S and ACE2, measured by Western Blot. 293T cells were transfected with or without IFITM1, TMPRSS2, S or ACE2 plasmids, and analyzed 18h later.


Impact of TMPRSS2 on S levels . D . TMPRSS2 decreases S surface levels , measured by flow cytometry . 293T cells were transfected with S plasmid , with or without TMPRSS2 , and analyzed 18h later . Murine polyclonal anti - S antibodies , one serum from a convalescent COVID - 19 patient ( sera n ° 111 ) , and two monoclonal antibodies ( anti - S1 and anti - S2 ) were tested . Data are representative of three independent experiments .

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Impact of TMPRSS2 on S levels. D. TMPRSS2 decreases S surface levels, measured by flow cytometry. 293T cells were transfected with S plasmid, with or without TMPRSS2, and analyzed 18h later. Murine polyclonal anti-S antibodies, one serum from a convalescent COVID-19 patient (sera n°111), and two monoclonal antibodies (anti-S1 and anti-S2) were tested. Data are representative of three independent experiments.


A Deletion of siaA , siaB , siaC , or siaD regulates biofilm formation . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down )

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A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down)


B The intracellular levels of c - di - GMPin PAO1 , ΔsiaC , ΔsiaB and the corresponding complemented strains were detected by LC - MS / MS

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B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS


E Bacterial two - hybrid assay reveals an interaction between SiaC and SiaD . The recombinant strains harboring different vectors were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P was used as positive controls

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E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls


F Pull down assays confirmed the interaction between SiaC and SiaD . Cell lysates of P . aeruginosa containing pMM67EH - siaD - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads

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F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads


G SPR measurements of SiaC ( left ) or SiaCP ( right ) binding at varying concentrations to SiaD . SiaC specifically interacted with SiaD with a KD of 24 . 7nM , whereas SiaCP failed to interact with SiaB . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant

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G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant


H Production of c - di - GMP by either SiaD or SiaD with SiaC at the indicated time - point was determined by HPLC

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H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC


A Bacterial two - hybrid assay reveals an interaction between SiaB and SiaC . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P were used as positive controls .

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A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls.


B Pull - down assays confirmed the interaction between SiaB and SiaC . Cell lysates of P . aeruginosa containing pMM67EH - siaC - Flag were incubated with GST or GST - SiaB individually , and protein complexes were captured by glutathione beads .

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B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads.


C SPR measurements of SiaC binding at varying concentrations to SiaB . SiaC specifically interacted with SiaB with a KD of 40 . 1 nM . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant .

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C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.


D SiaB - mediated regulation of biofilm formation is dependent on SiaC . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E Bacterial two - hybrid assayreveals an interaction between SiaC and SiaA PP2C - like domain . NA represents the empty vector pBT or pTRG . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . The strain expressing LGF2 and Gal11P were used as positive controls .

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E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls.


F Pull - down assays confirmed the interaction between SiaC and SiaA . Cell lysates of P . aeruginosa containing pMM67EH - siaA - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads .

F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


G Pull - down assays confirmed the interaction between SiaC and SiaA PP2C - like domain . Cell lysates of P . aeruginosa containing pMMB67EH - siaA386 - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads .

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G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


H The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate . Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides .

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B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides.


C Determination of modification sites by collision induced dissociation ( CID ) analysis . The MS / MS spectrum of modified L63LYLNTSSIK72 is shown . The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation , suggesting phosphorylation of Thr68 .

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C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68.


D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro . SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP . Phosphorylated products were separated by SDS - PAGE .

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D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE.


E Over - expression of SiaCT68A restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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E Over-expression of SiaCT68A restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


G Production of c - di - GMP at the indicated time - point by SiaD with SiaC or SiaCT68A was determined by HPLC . To evaluate the effect of SiaB - mediated phosphorylation on SiaD DGC activity , SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation .

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G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation.


C SiaCE33A partially restored the biofilm formation of ΔsiaA . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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C SiaCE33A partially restored the biofilm formation of ΔsiaA. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


D Triple mutation of E61A - Q64A - R67A is important for the function of SiaB . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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D Triple mutation of E61A-Q64A-R67A is important for the function of SiaB. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E Leu110 and Phe174 residues are important for SiaB function . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . Data represent the means and SDs of three biological replicates . * * , P < 0 . 01 based on one - way ANOVA test .

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E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test.


A - C ARR1 / 10 / 12 protein levels were examined by western blot when five - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , and 35S : ARR12 : YFP were treated with or without 200 mM NaCl for the indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - F Quantification analysis of the protein levels of ARR1 / 10 / 12 in ( A - C ) .

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A-C ARR1/10/12 protein levels were examined by western blot when five-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP were treated with or without 200 mM NaCl for the indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-F Quantification analysis of the protein levels of ARR1/10/12 in (A-C).


C In vivo Coimmunoprecipitation assay of MPK3 / 4 / 6 and ARR1 / 10 / 12 . MPK3 / 4 / 6 - YFP and ARR1 / 10 / 12 - MYC were co - expressed in Arabidopsis protoplast cells . Protein extracts ( Input ) were immunoprecipitated with GFP Trap magnetic agarose beads . Immunoblots were performed with anti - MYC antibody to detect ARR1 / 10 / 12 and with anti - GFP antibody to detect MPK3 / 4 / 6 .

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C In vivo Coimmunoprecipitation assay of MPK3/4/6 and ARR1/10/12. MPK3/4/6-YFP and ARR1/10/12-MYC were co-expressed in Arabidopsis protoplast cells. Protein extracts (Input) were immunoprecipitated with GFP Trap magnetic agarose beads. Immunoblots were performed with anti-MYC antibody to detect ARR1/10/12 and with anti-GFP antibody to detect MPK3/4/6.


A - C Thiophosphorylation assays of wild type and mutated forms ARR1 / 10 / 12 by MPK3 / 6 in vitro . Potential phosphorylated Ser residues or Thr residues of ARR1 , ARR10 , and ARR12 were mutated to Ala ( ARR1m / ARR10m / ARR12m ) . Recombinant ARR1 / 10 / 12 - His and ARR1m / 10m / 12m - His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD . Phosphorylated ARR1 / 10 / 12 , ARR1m / 10m / 12m , and MPK3 / 6 were visualized by an anti - thiophosphate ester - specific antibody ( α - TPE ) ( upper panel ) . Asterisk in ( B ) indicates non - specific bands . Recombinant MKK5DD , MPK3 , MPK6 , ARR1 / 10 / 12 , and ARR1m / 10m / 12m were detected by comassie brilliant blue ( CBB ) staining as loading controls ( bottom panel ) .

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A-C Thiophosphorylation assays of wild type and mutated forms ARR1/10/12 by MPK3/6 in vitro. Potential phosphorylated Ser residues or Thr residues of ARR1, ARR10, and ARR12 were mutated to Ala (ARR1m/ARR10m/ARR12m). Recombinant ARR1/10/12-His and ARR1m/10m/12m-His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD. Phosphorylated ARR1/10/12, ARR1m/10m/12m, and MPK3/6 were visualized by an anti-thiophosphate ester-specific antibody (α-TPE) (upper panel). Asterisk in (B) indicates non-specific bands. Recombinant MKK5DD, MPK3, MPK6, ARR1/10/12, and ARR1m/10m/12m were detected by comassie brilliant blue (CBB) staining as loading controls (bottom panel).


A - C Immunoblot analysis with anti - MYC antibody or anti - GFP antibody showing the upshift of ARR1 , ARR10 , and ARR12 in a phos - tag gel . The phosphorylated ARR1 , ARR10 , and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase ( CIAP ) . Seven - day - old seedlings of 35S : ARR1 : MYC ( A ) , 35S : ARR10 : YFP ( B ) , and 35S : ARR12 : YFP ( C ) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Total proteins were extracted and subjected to immunoblot analysis . ARR1 , ARR10 , and ARR12 proteins separated on SDS - PAGE gel without phos - tag were detected as loading controls .

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A-C Immunoblot analysis with anti-MYC antibody or anti-GFP antibody showing the upshift of ARR1, ARR10, and ARR12 in a phos-tag gel. The phosphorylated ARR1, ARR10, and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase (CIAP). Seven-day-old seedlings of 35S:ARR1:MYC (A), 35S:ARR10:YFP (B), and 35S:ARR12:YFP (C) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Total proteins were extracted and subjected to immunoblot analysis. ARR1, ARR10, and ARR12 proteins separated on SDS-PAGE gel without phos-tag were detected as loading controls.


A - C Immunoblot analysis the effects of MPK3 / 6 on salt stress - induced ARR1 / 10 / 12 protein degradation . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , and 35S : ARR12 : YFP in Col - 0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I Quantification analysis of MPK3 / 6 effect on salt stress - induced ARR1 / 10 / 12 protein degradation in ( A - C ) .

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A-C Immunoblot analysis the effects of MPK3/6 on salt stress-induced ARR1/10/12 protein degradation. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP in Col-0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of MPK3/6 effect on salt stress-induced ARR1/10/12 protein degradation in (A-C).


A - C Immunoblot analysis stability of ARR1 / 10 / 12 and ARR1T553A / 10S383A / 12S323A proteins under salt stress . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP , 35S : ARR1T553A : MYC , 35S : ARR10S383A : YFP , and 35S : ARR12S323A : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I Quantification analysis of stability of ARR1 / 10 / 12 and ARR1T553A / 10S383A / 12S323A proteins under salt stress in ( A - C ) .

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A-C Immunoblot analysis stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, 35S:ARR12:YFP, 35S:ARR1T553A:MYC, 35S:ARR10S383A:YFP, and 35S:ARR12S323A:YFP were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress in (A-C).


Four - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR1T553A : MYC , and 35S : ARR1T553D : MYC were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes ( A ) were determined .

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Four-day-old seedlings of 35S:ARR1:MYC, 35S:ARR1T553A:MYC, and 35S:ARR1T553D:MYC were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Four - day - old seedlings of 35S : ARR10 : YFP , 35S : ARR10S383A : YFP , and 35S : ARR10S383D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes ( A ) were determined .

Four-day-old seedlings of 35S:ARR10:YFP, 35S:ARR10S383A:YFP, and 35S:ARR10S383D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Four - day - old seedlings of 35S : ARR12 : YFP , 35S : ARR12S323A : YFP , and 35S : ARR12S323D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes were determined .

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Four-day-old seedlings of 35S:ARR12:YFP, 35S:ARR12S323A:YFP, and 35S:ARR12S323D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes were determined.


Three - day - old seedlings of Col - 0 , MPK3SR , MPK3SRarr1 / 12 , and arr1 / 12 were transferred to 0 . 5 μM NA - PP1 - containing 1 / 2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1 / 12 and then the plants were transferred to 0 . 2 μM NA - PP1 - containing 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . Pictures were taken and the elongated root length was determined 3 d

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Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later


A - C . Positioning of Upf1 , Upf2 and Upf3 in the abundance distribution of all yeast proteins , based on the data compiled by Ho et al . , 2018 ( A )

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A-C. Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al., 2018 (A)


The distribution of abundance for the proteins identified by mass spectrometry in input ( B ) show the expected bias for proteins present in high copy

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The distribution of abundance for the proteins identified by mass spectrometry in input (B) show the expected bias for proteins present in high copy numbers


The distribution of abundance for the proteins identified by mass spectrometry i purified samples ( C ) , show the expected bias for proteins present in high copy numbers . Light grey colour highlights the positions of proteins that were only identified in the Upf1 - TAP samples ( 6 replicates ) and not in the total extract ( 3 replicates )

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The distribution of abundance for the proteins identified by mass spectrometry i purified samples (C), show the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1-TAP samples (6 replicates) and not in the total extract (3 replicates)


D . Correlation of LTOP2 - based protein level estimates in a total yeast protein extract with published protein abundance data ( Ho et al , 2018 )

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D. Correlation of LTOP2-based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018)


E . Distribution of the intensity for the proteins quantified in association with Upf1 ( LTOP2 score , log2 scale ) . Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red . The dark grey line indicates the region of intensities corresponding to ribosomal proteins F . Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract . Upf1 , Upf2 and Upf3 positions are also highlighted as white rectangles G . A combination of the data presented in ( E ) and ( F ) as a scatter plot , to see both the amount and enrichment of proteins in Upf1 - TAP purified samples . The horizontal axis represents the LTOP2 score , with the vertical axis showing enrichment over input values . Both axes use log2 transformed values

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E. Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log2 scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins F. Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white rectangles G. A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1-TAP purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log2 transformed values


A - F . Enrichment values for proteins identified in tagged Upf1 ( A ) , Upf2 ( B ) , Upf3 ( C ) , Nmd4 ( D ) , Ebs1 ( E ) , and Dcp1 ( F ) purifications . Tagged proteins are indicated in bold for each experiment . Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment . Error bars represent SD . Dots represent individual enrichment values for each protein in the 6 replicates for Upf1 - TAP , 3 for Upf1 - TAP and RNase , 6 for Upf2 - TAP , 3 for Upf2 - TAP and RNase , 5 for Upf3 - TAP , 3 for Upf3 - TAP and RNase , 4 for Nmd4 - TAP , 3 for Nmd4 - TAP and RNase , 3 for Ebs1 - TAP , with and without RNase , and 3 for Dcp1 - TAP with or without RNase . Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown . For clarity , groups of related proteins were combined ( the group of Lsm1 to Lsm7 is marked as " Lsm1 - 7 " and the values correspond to the mean of all the values found in purifications ; CK2 group corresponds to Cka1 , Cka2 , Ckb1 and Ckb2 )

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A-F. Enrichment values for proteins identified in tagged Upf1 (A), Upf2 (B), Upf3 (C), Nmd4 (D), Ebs1 (E), and Dcp1 (F) purifications. Tagged proteins are indicated in bold for each experiment. Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment. Error bars represent SD. Dots represent individual enrichment values for each protein in the 6 replicates for Upf1-TAP, 3 for Upf1-TAP and RNase, 6 for Upf2-TAP, 3 for Upf2-TAP and RNase, 5 for Upf3-TAP, 3 for Upf3-TAP and RNase, 4 for Nmd4-TAP, 3 for Nmd4-TAP and RNase, 3 for Ebs1-TAP, with and without RNase, and 3 for Dcp1-TAP with or without RNase. Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown. For clarity, groups of related proteins were combined (the group of Lsm1 to Lsm7 is marked as "Lsm1-7" and the values correspond to the mean of all the values found in purifications; CK2 group corresponds to Cka1, Cka2, Ckb1 and Ckb2)


G . Immunoblot validation of Upf1 - TAP interactions with HA tagged Nmd4 and Ebs1 . Control strains did not express Upf1 -

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G. Immunoblot validation of Upf1-TAP interactions with HA tagged Nmd4 and Ebs1. Control strains did not express Upf1-TAP


H . Immunoblot validation of Upf1 - TAP interaction with Pat1 - HA , Lsm1 - HA and Edc3 - HA . The purification was performed with a mix of three strains , expressing Upf1 - TAP or not ( control )

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H. Immunoblot validation of Upf1-TAP interaction with Pat1-HA, Lsm1-HA and Edc3-HA. The purification was performed with a mix of three strains, expressing Upf1-TAP or not (control)


B - D . Results of purifications using Upf1 - FL ( B ) , Upf1 - CH ( C as bait with the x - axis showing the average enrichment value ( log2 scale ) for Upf1 - decapping and Upf1 - 23

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B-D. Results of purifications using Upf1-FL (B), Upf1-CH (C as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components


B - D . Results of purifications usin Upf1 - Cter ( D ) as bait with the x - axis showing the average enrichment value ( log2 scale ) for Upf1 - decapping and Upf1 - 23 components . The colour of the bars illustrates the treatment used during the purification , black without RNase , and orange with an RNase treatment . Error bars represent SD . White dots correspond to enrichment values obtained in individual

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B-D. Results of purifications usin Upf1-Cter (D) as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase, and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments


E . Comparison of the enrichment of Upf1 , Nmd4 and Ebs1 in the purification of the Upf1 fragments . The Upf1 C - terminal region ( 854 - 971 ) affected the association with Nmd4 or Ebs1 ( Student t - test with a one - sided alternative hypothesis ) . We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region ( Upf1 - FL and Upf1 - HD - Cter , 6 experiments ) and purifications of Upf1 fragments lacking the extension ( Upf1 - CH - HD and Upf1 - HD , 4 experiments ) . The C - terminal region had no effect on Nmd4 enrichment ( p - value ≈ 0 . 98 ) , whereas there was significantly less associated Ebs1 on this small region ( p - value ≈ 0 . 0013 )

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E. Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C-terminal region (854-971) affected the association with Nmd4 or Ebs1 (Student t-test with a one-sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1-FL and Upf1-HD-Cter, 6 experiments) and purifications of Upf1 fragments lacking the extension (Upf1-CH-HD and Upf1-HD, 4 experiments). The C-terminal region had no effect on Nmd4 enrichment (p-value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (p-value ≈ 0.0013)


F . Western blot of input and eluates of Upf1 domains purification in a Nmd4 - HA strain . The band with the # might corresponds to a dimer of Upf1 - CH , bands marked with a star correspond to residual signal with the anti - HA antibodies ( Nmd4 ) . Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease . G6PDH served as a loading control in the input

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F. Western blot of input and eluates of Upf1 domains purification in a Nmd4-HA strain. The band with the # might corresponds to a dimer of Upf1-CH, bands marked with a star correspond to residual signal with the anti-HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples


A . Average enrichment of Upf1 - 23 and Upf1 - decapping components in purified Upf1 - TAP in the presence ( black bars ) and absence ( blue bars ) of NMD4 . Pink and dark blue vertical lines highlight proteins of the Upf1 - 23 and Upf1 - decapping complexes respectively . White dots correspond to individual enrichment values obtained in replicate experiments , 6 for Upf1 - TAP and 3 for the nmd4Δ condition . Error bars correspond to SD

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A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD


B . Average enrichment of Upf1 - decapping components in Nmd4 - TAP in the presence ( black bars ) and absence ( blue bars ) of UPF1 . White dots correspond to individual enrichment values obtained in replicate experiments , 4 for Nmd4 - TAP and 2 for the upf1Δ condition . Error bars correspond to SD

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B. Average enrichment of Upf1-decapping components in Nmd4-TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4-TAP and 2 for the upf1Δ condition. Error bars correspond to SD


D . CBP - Nmd4 was mixed with hUPF1 - HD or yUPF1 - HD ( all the proteins overexpressed in E . coli and purified ) . Protein mixtures before ( input , 20 % of total ) or after purification on calmodulin affinity beads were separated on 10 % SDS - PAGE ( w / v ) acrylamide gels

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D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels


E . Upf1 interaction with a 30 - mer 3 ' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution

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E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution


B . Scatter plot of the mean fold change of transcripts in nmd4 ∆ RNAseq experiment against transcript mean fold change in ebs1 ∆ ( Pearson correlation , r = 0 . 66 , p - value < 2 . 10 - 16 ) . Black dots correspond to NMD substrate examples for which individual traces are shown in Fig . EV3 . Dashed lines represent boundaries of five bins of equal numbers of transcripts ( 2046 transcripts or notable features per bin ) , with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild

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B. Scatter plot of the mean fold change of transcripts in nmd4∆ RNAseq experiment against transcript mean fold change in ebs1∆ (Pearson correlation, r = 0.66, p-value < 2.10-16). Black dots correspond to NMD substrate examples for which individual traces are shown in Fig. EV3. Dashed lines represent boundaries of five bins of equal numbers of transcripts (2046 transcripts or notable features per bin), with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild type


C . Percentage of transcripts affected by UPF1 deletion ( increase by at least 1 . 4 fold ) among nmd4 ∆ bins , as defined in B . Differences between percentages of upf1 ∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant ( binomial test , p < 10 - 24 ) . D . Same as in C , for ebs1 ∆ . Differences between distribution of transcripts in bin n and bin n + 1 were significant ( binomial test , p < 10 - 9 )

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C. Percentage of transcripts affected by UPF1 deletion (increase by at least 1.4 fold) among nmd4∆ bins, as defined in B. Differences between percentages of upf1∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant (binomial test, p<10-24). D. Same as in C, for ebs1∆. Differences between distribution of transcripts in bin n and bin n+1 were significant (binomial test, p<10-9)


E . NMD efficiency of WT , upf1 ∆ or double mutant strains complemented with Upf1 - FL , Upf1 - HD - Cter or an empty plasmid . The NMD efficiency for each strain is based on reverse - transcription followed by quantitative PCR for RPL28 pre - mRNA . A wild - type strain has 100 % NMD efficiency and a upf1 ∆ strain has 0 % NMD efficiency , by definition . Each value is an average of replicate experiments , 3 for the WT condition , 5 for upf1Δ , 3 for ufp1Δ / upf2Δ , upf1Δ / ufp3Δ and upf1Δ / ebs1Δ and 4 for the upf1Δ / nmd4Δ condition . Error bars represent SD . Dots represent values obtained in the various replicate

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E. NMD efficiency of WT, upf1∆ or double mutant strains complemented with Upf1-FL, Upf1-HD-Cter or an empty plasmid. The NMD efficiency for each strain is based on reverse-transcription followed by quantitative PCR for RPL28 pre-mRNA. A wild-type strain has 100% NMD efficiency and a upf1∆ strain has 0% NMD efficiency, by definition. Each value is an average of replicate experiments, 3 for the WT condition, 5 for upf1Δ, 3 for ufp1Δ/upf2Δ, upf1Δ/ufp3Δ and upf1Δ/ebs1Δ and 4 for the upf1Δ/nmd4Δ condition. Error bars represent SD. Dots represent values obtained in the various replicate experiments


G . Changes in the steady - state level of the reporter encoded HA - tagged protein in upf1Δ , nmd4Δ , ebs1Δ and the nmd4Δ / ebs1Δ strains were estimated by immunoblot from three independent experiments , with one example

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G. Changes in the steady-state level of the reporter encoded HA-tagged protein in upf1Δ, nmd4Δ, ebs1Δ and the nmd4Δ/ebs1Δ strains were estimated by immunoblot from three independent experiments, with one example shown


H . RNA decay in a wild - type and nmd4Δ / ebs1Δ strains was tested by reverse - transcription quantitative PCR specific to the 5 " end of the reporter RNA at different times after transcription shut - off . The quantifications represent mean RNA amounts and standard deviation of the average ( three independent

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H. RNA decay in a wild-type and nmd4Δ/ebs1Δ strains was tested by reverse-transcription quantitative PCR specific to the 5" end of the reporter RNA at different times after transcription shut-off. The quantifications represent mean RNA amounts and standard deviation of the average (three independent experim


A - B . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf1 - TAP purified samples in the presence ( grey bars ) or absence ( blue bars ) of UPF2 ( AError bars represent SD . White dots represent enrichment values for individual replicates , 6 for Upf1 - TAP , 3 for the upf2Δ an

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A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1 -TAP purified samples in the presence (grey bars) or absence (blue bars) of UPF2 (AError bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for the upf2Δ an conditions


A - B . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf1 - TAP purified samples in the presence ( grey bars ) or absence ( blue bars ) o UPF3 ( B ) . Error bars represent SD . White dots represent enrichment values for individual replicates , 6 for Upf1 - TAP , 3 for th upf3Δ

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A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1-TAP purified samples in the presence (grey bars) or absence (blue bars) o UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for th upf3Δ conditions


C - D . Evaluation of the effects of UPF2 deletion on Upf3 - TAP levels ( C in total extracts , in comparison with a loading control ( G6PDH ) was done by

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C-D. Evaluation of the effects of UPF2 deletion on Upf3-TAP levels (C in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot


C - D . Evaluation of the effect of UPF3 deletion on Upf2 - TAP levels ( D ) in total extracts , in comparison with a loading control ( G6PDH ) was done by

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C-D. Evaluation of the effect of UPF3 deletion on Upf2-TAP levels (D) in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot


E . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf2 - TAP purified samples in the presence ( grey bars , 6 experiments ) or absence ( blue bars , 2 experiments ) of UPF1 . Error bars represent SD . The levels of expression of Upf2 - TAP and Upf3 - TAP proteins have been verified in this condition ( Appendix Fig . S2 )

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E. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf2-TAP purified samples in the presence (grey bars, 6 experiments) or absence (blue bars, 2 experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2-TAP and Upf3-TAP proteins have been verified in this condition (Appendix Fig. S2)


A . Unspliced RPL28 , an NMD substrate , was enriched in Nmd4 - TAP purification in comparison with a control untagged strain , as measured by reverse transcription followed by quantitative PCR . Bars correspond to the mean of pre - RPL28 enrichment for 3 independent experiments , as compared with RIM1 , a non - NMD mRNA . Error bars correspond to SD . The indicated p - value was computed using the Welch t - test , single - sided comparison

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A. Unspliced RPL28, an NMD substrate, was enriched in Nmd4-TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre-RPL28 enrichment for 3 independent experiments, as compared with RIM1, a non-NMD mRNA. Error bars correspond to SD. The indicated p-value was computed using the Welch t-test, single-sided comparison


B - D . Distribution of Nmd4 - TAP , Nog1 ( marker of free 60S subunits ) and Rps8 ( marker of 40S , 80S and polysome fractions ) in wild type ( B ) , upf1 ∆ ( C ) and upf2 ∆ ( D ) strains , tested by immunoblot . Fractions 1 , 2 , 3 are light fractions , fractions 4 , 5 and 6 correspond respectively to ribosomal 40S , 60S and 80S positions , fractions 7 to 12 correspond to polysomes . The percent of total signal for Nmd4 - TAP in three independent replicates along with standard deviation of the values are indicated for each

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B-D. Distribution of Nmd4-TAP, Nog1 (marker of free 60S subunits) and Rps8 (marker of 40S, 80S and polysome fractions) in wild type (B), upf1∆ (C) and upf2∆ (D) strains, tested by immunoblot. Fractions 1, 2, 3 are light fractions, fractions 4, 5 and 6 correspond respectively to ribosomal 40S, 60S and 80S positions, fractions 7 to 12 correspond to polysomes. The percent of total signal for Nmd4-TAP in three independent replicates along with standard deviation of the values are indicated for each fraction


E . Quantified relative changes in Nmd4 - TAP average levels in light and monosome / polysome fractions in upf1 ∆ and upf2 ∆ strains ( 100 % correspond to levels in the wild type strain ) . Indicated p - values correspond to the Welch t - test , single - sided comparison of 4 replicate experiments ( individual values are indicated as dots ) . Error bars correspond to SD

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E. Quantified relative changes in Nmd4-TAP average levels in light and monosome/polysome fractions in upf1∆ and upf2∆ strains (100% correspond to levels in the wild type strain). Indicated p-values correspond to the Welch t-test, single-sided comparison of 4 replicate experiments (individual values are indicated as dots). Error bars correspond to SD


a ) The number of somatic mutations discovered from each paired NPC case subjected to WES and key clinical parameters ( Supplementary Table 2a ) clustered according to the clinical stage . Indel , insertion or deletion . ( b ) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC . The font size of the nucleotides at the 3 ′ and 5 ′ positions is proportional to their frequencies ( Supplementary Fig . 3b ) .

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a) The number of somatic mutations discovered from each paired NPC case subjected to WES and key clinical parameters (Supplementary Table 2a) clustered according to the clinical stage. Indel, insertion or deletion. (b) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC. The font size of the nucleotides at the 3′ and 5′ positions is proportional to their frequencies (Supplementary Fig. 3b).