C , D Whole blood was either treated with 24F4A for 1 h at 37 ° C ( pre - incubation ) or left untreated . PBMC were isolated from each condition . PBMC from untreated whole blood were subsequently treated with 10 μg / ml 24F4A , from conditions were stimulated with CpG - A for 16 h . Plot represents mean of duplicate wells . Flow cytometry was used to measure BDCA2 ( MFI ) . Shown is a representative donor of eight donors tested .

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C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.


E Whole blood was pre - treated for 0 , 1 , 3 , 6 , 9 h with 10 μg / ml of 24F4 or isotype control then stimulated with CpG - A for additional 16 h . IFNα were measured by ELISA . Shown is a representative plot of two independent experiments .

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E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . A - C Prior to in vivo dosing , baseline surface of BDCA2 for both vehicle ( Ai ) 1 mg / kg ( Bi ) ( red , dotted ) were established by with fluorescently labeled 24F4A ( direct method ) . Maximal binding of 24F4A to BDCA2 was also established pre - dose in vehicle ( Aii ) 1 mg / kg ( Bii ) ( red , solid ) by treating whole blood with 10 μg / ml of 24F4A at 4 ° C then detecting bound 24F4A with a fluorescently labeled anti - human IgG1 ( indirect method ) . direct method was used to stain whole blood from both vehicle ( Aiii ) 1 mg / kg 24F4A ( Biii ) 6 h post - dose ( red , dotted ) . In a separate stain , indirect method was used to detect bound 24F4A in vehicle ( Aiv ) 1 mg / kg ( Biv ) treated ( black , solid ) . ( C ) BDCA2 internalization relative to pre - dose BDCA2 6 h post - dose with vehicle , 10 mg / kg , or 1 mg / kg 24F4A . Graph shows mean ± standard deviation for each group ( n = 3 ) .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . D PK / PD relationship between 24F4Aserum concentrations ( red triangle , left axis ) BDCA2 expression on pDCs ( black squares , right axis , normalized to pre - dose ) from 1 mg / kg group ( i - iii ) . Serum 24F4A was measured by ELISA . ( iv ) BDCA2 internalization versus serum of 24F4A for dosed cynomolgus monkeys at time points tested .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . E Whole blood from vehicle - or 1 mg / kg 24F4A - treated monkeys was stimulated with CpG - A , induction of IFN - I was measured by MxA bioassay at various time points pre - post - treatment . Horizontal black lines represent model - estimates of geometric mean of IFN - I in pre - post - dose samples . Duplicate symbols represent independent replicates of MxA bioassay for that time point . Statistical was performed using a two - way mixed - effects of variance ( ANOVA ) .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).


A - F Isolated human pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( black squares ) , or 10 μg / ml of isotype control ( black triangle ) stimulated with 5 μM of R848 ( A B ) , 1 μM of CpG - A ( C D ) , or immune complexes ( E F ) for 16 h at 37 ° C . IFNα concentrations in cultured supernatants were determined using ELISA . Representative plots for each stimulation ( A , C E ) average IC50 of 24F4A 24F4A - ef for each stimulation condition ( B , D F ) are shown ( n = 3 ) . Horizontal bars represent mean IC50 , error bars represent SD of mean from three independent experiments .

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A-F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.


G Isolated human pDCs were treated with 10 μg / ml of 24F4A ( black ) , 24F4A - ef ( gray shaded histogram ) , or isotype control ( gray dotted ) for 10 min at 37 ° C . Flow cytometry was used to evaluate pSYK pPLCγ2 . Shown is a representative plot of two independent experiments .

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G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.


H Isolated pDCs treated with 10 μg / ml of anti - CD32a ( AT10 ) stimulated with CpG - A , R848 , or immune complexes . Error bars represent SD of inhibition of IFNα from three independent experiments .

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H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.


A - F Isolated pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( squares ) , Chi - 6G6 ( triangle ) , or isotype control ( diamond ) for 16 h at 37 ° C stimulated with CpG - A . Flow cytometry was performed to evaluate MFI of ( A ) BDCA2 ( C ) CD32a on surface of pDCs . internalization of BDCA2 ( B ) CD32a ( D ) on pDCs treated with 10 μg / ml of 24F4A , 24F4A - ef , Chi - 6G6 , or isotype control . Error bars represent SD of internalization of five experiments conducted . ( E ) CD32a downmodulation on isolated pDCs treated with 10 μg / ml of an anti - CD40 mAb . Error bars represent SD of downmodulation of two independent experiments . FMO represents background of CD32a ( inverted triangle ) . ( F ) of maximal expression of BDCA2 or CD32a in presence of increasing concentrations of 24F4A or Chi - 6G6 . Maximal expression was defined by MFI of both BDCA2 CD32a on untreated . Shown is a representative plot of five independent experiments conducted .

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A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.


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

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

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

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

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


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

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

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(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 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 intensities at last time point were determined by single exponential fitting . error bars represent median interquartile range ( N = 20 for each condition ) . Mann - Whitney U ( two - ) was used to obtain a P value .

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

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

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

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

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(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 structure of NuMA microtubules upon centrosome removal . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were observed with a 60 × objective . Red green represent NuMA 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 time of NEBD .

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(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 were treated with 100 ng / ml nocodazole . Gray , red , green blue represent α - tubulin , NuMA , CEP152 ( mClover ) , respectively . Scale bar , 5 μm .

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

O O O O 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 were treated with 1 μg / ml doxycycline ( Dox ) 500 μM indole - 3 - acetic acid ( IAA ) . Arrowhead indicates spindle pole . Green , red , gray blue represent DHC1 ( mClover ) , NuMA , GT335 , 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 B-CONTROLLED_VAR I-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 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 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 ) distribution of NuMA upon DHC1 depletion . Arrowheads indicate presence of NuMA on microtubules . Green , red , blue represent α - tubulin , NuMA , , 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 assembled NuMA structure . Green , red , gray blue represent endogenous NuMA ( mClover ) , expressed NuMA ( mCherry ) , GT335 , respectively . Z - projections of 21 sections , every 1 μm . Scale bar , 5 μm . ( H ) 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 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 B-MEASURED_VAR 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 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

(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 spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with 1 μg / ml doxycycline ( Dox ) 500 μM IAA . Two - way arrow indicates bipolarity . Gray , green , red , blue represent α - tubulin , NuMA ( mClover ) , GT335 , , 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 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 B-CONTROLLED_VAR 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 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 ) localization of Eg5 in centrinone - treated acentrosomal HeLa . Red , gray , green blue represent NuMA , Eg5 , GT335 , 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 ) localization of Eg5 in spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with Dox 500 μM IAA . Gray , green , red , blue represent Eg5 , NuMA ( mClover ) , CP110 , , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

O O O O O B-MEASURED_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 B-CONTROLLED_VAR 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

(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 spindle upon Eg5 inhibition . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with 30 μM monastrol . Gray , green , red , blue represent α - tubulin , NuMA ( mClover ) , GT335 , , 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 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 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 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 establishment of bipolarity within acentrosomal spindle pole upon inhibition of Eg5 . HeLa expressing EGFP - centrin1 mCherry - NuMA were observed in presence of 50 μM of monastrol with a 63 × objective . Magenta green represent mCherry - NuMA EGFP - centrin1 , respectively . Arrowheads two - way arrows indicate monopolar bipolar states , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) time of bipolarity establishment being stuck in monopolar - like state of acentrosomal spindle poles in ( A ) . Each plot shows cumulative of each event at each time point ( N = total 75 from two independent experiments ) . (

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 dynamics of mClover - NuMA chromosomes . HeLa expressing mClover - NuMA were observed in presence of SiR - with a 63 × objective . Magenta green represent SiR - mClover - NuMA , respectively . Arrowheads indicate initial bipolarity in acentrosomal spindle pole ( 8 min ) , dispersed chromosomes ( 328 min ) , monopolar - like state of 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 ) time of bipolarity establishment being stuck in monopolar - like state of acentrosomal spindle poles in ( C ) . Each plot shows cumulative of each event at each time point ( N = total 35 from three independent experiments ) .

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 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

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

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

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

O O O O O B-MEASURED_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 B-CONTROLLED_VAR 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

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

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-CONTROLLED_VAR O O O O O O O O O O O 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 O O O O O O O O O O O O O O O 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) 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 normal groups . Rows represent circRNAs , columns represent samples . Rows were ordered by fold change , columns were ordered by their group . sample of N8 was not included due to low sequencing library .

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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 recurrence nonrecurrence groups . Both row column were unsupervised clustered with 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 . 4 of 22 differentially expressed circRNAs were confirmed by qRT - PCR , which were retained after marker selection procedure . * * P < 0 . 01 , Student ' s t - , 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 of circle cirScore for predicting recurrence in training dataset . P - values are shown for indicated comparison of AUC between each marker cirScore . Student ' s t - , AUC = under 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 ) OS ( B ) in 249 in training set . Kaplan - Meier curves of DFS ( C ) OS ( D ) in 122 in internal validation data set . Kaplan - Meier curves of DFS ( E ) OS ( F ) in 180 in external independent validation data set . Hazard ratios ( HRs ) were calculated with a univariate Cox regression , P - values were calculated with log - rank .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 convergent primers showing circularization of has _ circ _ 0079480 has _ circ _ 0087391 . cDNA , complementary ; gDNA , genomic .

O O O O O O O O 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. 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 knockdown efficiency of has _ circ _ 0079480 has _ circ _ 0087319 in SW620 HCT116 transfected with two unique shRNAs ( # 1 , # 2 ) . * * P < 0 . 01 , Student ' s t - , 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 of migration phenotype in HCT116 with knockdown of candidate circRNAs .

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


D . relative fold - change of transwell migration for indicated knockdown over those of control . * * P < 0 . 01 , Student ' s t - , 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 eosin ( H & E ) statistical results of micro - metastatic nodules in liver from mice injected with indicated into spleen for 45 days . N = 8 per group . * * P < 0 . 01 , Student ' s t - , 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 statistical results of metastatic lung nodules from mice injected with indicated via tail vein for 60 days . ( Five sections evaluated per lung ) . N = 8 per group . * * P < 0 . 01 , Student ' s t - , 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 of GFP ( syncytia ) Propidium Iodide ( PI ) ( ) 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 . of U2OS - ACE2 fusion by time - lapse microscopy . Results are mean ± sd from 3 fields per condition .

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


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

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


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

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


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


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


were infected at indicated multiplicity of infection ( MOI ) analyzed after 20h . C . fusion of IFITM1 + with U2OS - ACE2 syncytia is drastically reduced . Right panel : 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


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

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

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 of - fusion . Scale bar : 100 µm .

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


C . 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 . were monitored by video microscopy GFP 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 ) .

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

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 IFITMs on kinetics of fusion by S - expressing 293T . 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 ( Donor ) , ACE2 - expressing ( Acceptor ) were co - transfected with TMPRSS2 , IFITM or control plasmids . fusion was quantified by measuring GFP + after 18h . indicated combinations were tested . impact of IFITMs was measured in absence of TMPRSS2 ( left panel ) , in presence of TMPRSS2 in donor ( middle panel ) or acceptor ( right panel ) . Data are mean ± sd of 4 independent experiments . Statistical : 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 . B . TMPRSS2 does not decrease IFITM amounts measured by flow cytometry . 293T were transfected with indicated IFITM plasmids , with or without TMPRSS2 , 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 ACE2 , measured by Western Blot . 293T were transfected with or without IFITM1 , TMPRSS2 , S or ACE2 plasmids , 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 . D . TMPRSS2 decreases S surface , measured by flow cytometry . 293T were transfected with S plasmid , with or without TMPRSS2 , analyzed 18h later . Murine polyclonal anti - S antibodies , one serum from a convalescent COVID - 19 ( sera n ° 111 ) , two monoclonal antibodies ( anti - S1 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 . biofilm formation of indicated strains was displayed with crystal violet ( up ) 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 intracellular of c - di - GMPin PAO1 , ΔsiaC , ΔsiaB 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 two - hybrid reveals an interaction between SiaC SiaD . recombinant strains harboring different vectors were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . NA represents empty vector pBT or pTRG . expressing LGF2 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 interaction between SiaC SiaD . lysates of P . aeruginosa containing pMM67EH - siaD - Flag were incubated with GST or GST - SiaC individually , 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 ) 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 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 two - hybrid reveals an interaction between SiaB SiaC . recombinant strains harboring different were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . NA represents empty vector pBT or pTRG . expressing LGF2 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 interaction between SiaB SiaC . lysates of P . aeruginosa containing pMM67EH - siaC - Flag were incubated with GST or GST - SiaB individually , 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 ) 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 . biofilm formation of indicated strains was displayed with crystal violet ( up ) 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 two - hybrid assayreveals an interaction between SiaC SiaA PP2C - like domain . NA represents empty vector pBT or pTRG . recombinant strains harboring different were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . expressing LGF2 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 interaction between SiaC SiaA . lysates of P . aeruginosa containing pMM67EH - siaA - Flag were incubated with GST or GST - SiaC individually , 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 interaction between SiaC SiaA PP2C - like domain . lysates of P . aeruginosa containing pMMB67EH - siaA386 - Flag were incubated with GST or GST - SiaC individually , 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 biofilm formation of indicated strains was displayed with crystal violet ( up ) 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 doubly protonated peptide are shown with peak intensities indicating relative amount of either 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 ) . MS / MS spectrum of modified L63LYLNTSSIK72 is shown . fragment ions b6 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 SiaA dephosphorylates SiaC in vitro . SiaA or SiaAT68A were added to reactions containing SiaB or SiaA386 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 ability of ΔsiaA to form biofilm . biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) . F Deletion of ΔsiaB in ΔsiaA background restored ability of ΔsiaA to form biofilm . biofilm formation of indicated strains was displayed with crystal violet ( up ) 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 indicated time - point by SiaD with SiaC or SiaCT68A was determined by HPLC . To evaluate effect of SiaB - mediated phosphorylation on SiaD DGC activity , SiaC or SiaCT68A was pretreated by SiaB in 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 biofilm formation of ΔsiaA . biofilm formation by indicated strains was displayed with crystal violet ( up ) 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 function of SiaB . biofilm formation by indicated strains was displayed with crystal violet ( up ) 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 Phe174 residues are important for SiaB function . biofilm formation by indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) . Data represent means SDs of three biological replicates . * * , P < 0 . 01 on one - way ANOVA .

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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 were examined by western blot when five - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative 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 of 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 of MPK3 / 4 / 6 ARR1 / 10 / 12 . MPK3 / 4 / 6 - YFP ARR1 / 10 / 12 - MYC were co - expressed in Arabidopsis protoplast . extracts ( Input ) were immunoprecipitated with GFP Trap magnetic agarose beads . Immunoblots were performed with anti - MYC antibody to detect ARR1 / 10 / 12 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 mutated forms ARR1 / 10 / 12 by MPK3 / 6 in vitro . Potential phosphorylated Ser residues or Thr residues of ARR1 , ARR10 , ARR12 were mutated to Ala ( ARR1m / ARR10m / ARR12m ) . Recombinant ARR1 / 10 / 12 - His ARR1m / 10m / 12m - His were incubated with MPK3 MPK6 which were activated by MKK5DD . Phosphorylated ARR1 / 10 / 12 , ARR1m / 10m / 12m , 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 , ARR1m / 10m / 12m were detected by comassie brilliant blue ( CBB ) 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 with anti - MYC antibody or anti - GFP antibody showing upshift of ARR1 , ARR10 , ARR12 in a phos - tag gel . phosphorylated ARR1 , ARR10 , ARR12 were abolished after treatment with calf intestinal alkaline phosphatase ( CIAP ) . Seven - day - old seedlings of 35S : ARR1 : MYC ( A ) , 35S : ARR10 : YFP ( B ) , 35S : ARR12 : YFP ( C ) in Col MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Total were extracted subjected to immunoblot . ARR1 , ARR10 , ARR12 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 effects of MPK3 / 6 on salt stress - induced ARR1 / 10 / 12 degradation . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP in Col - 0 MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative 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 of MPK3 / 6 effect on salt stress - induced ARR1 / 10 / 12 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 stability of ARR1 / 10 / 12 ARR1T553A / 10S383A / 12S323A under salt stress . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP , 35S : ARR1T553A : MYC , 35S : ARR10S383A : YFP , 35S : ARR12S323A : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative 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 of stability of ARR1 / 10 / 12 ARR1T553A / 10S383A / 12S323A 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 , 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 , ( 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 , 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 , ( 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 , 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 , 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 , arr1 / 12 were transferred to 0 . 5 μM NA - PP1 - containing 1 / 2 MS medium for 2 d to inhibit MPK3TG in MPK3SR MPK3SRarr1 / 12 then 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 elongated root 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 Upf3 in abundance distribution of yeast , on 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)


distribution of abundance for identified by mass spectrometry in input ( B ) show expected bias for 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


distribution of abundance for identified by mass spectrometry i purified samples ( C ) , show expected bias for present in high copy numbers . grey colour highlights positions of that were only identified in Upf1 - TAP samples ( 6 replicates ) not in 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 - level estimates in a total yeast extract with published 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 for quantified in association with Upf1 ( LTOP2 score , log2 scale ) . Positioning of Upf1 is highlighted as a rectangle while Upf2 Upf3 are highlighted in red . grey indicates region of intensities corresponding to ribosomal F . Distribution of enrichment values for Upf1 associated on LTOP2 scores known abundance in a total yeast extract . Upf1 , Upf2 Upf3 positions are also highlighted as rectangles G . A combination of data presented in ( E ) ( F ) as a scatter plot , to see both amount enrichment of in Upf1 - TAP purified samples . horizontal axis represents LTOP2 score , with 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 identified in tagged Upf1 ( A ) , Upf2 ( B ) , Upf3 ( C ) , Nmd4 ( D ) , Ebs1 ( E ) , Dcp1 ( F ) purifications . Tagged are indicated in bold for each experiment . Black bars correspond to average enrichment values obtained in purifications done without RNase orange bars with an RNase A RNase T1 treatment . Error bars represent SD . Dots represent enrichment values for each in 6 replicates for Upf1 - TAP , 3 for Upf1 - TAP RNase , 6 for Upf2 - TAP , 3 for Upf2 - TAP RNase , 5 for Upf3 - TAP , 3 for Upf3 - TAP RNase , 4 for Nmd4 - TAP , 3 for Nmd4 - TAP RNase , 3 for Ebs1 - TAP , with without RNase , 3 for Dcp1 - TAP with or without RNase . Only enriched by a factor of 16 or more in one of purifications presented here are shown . For clarity , groups of related were combined ( group of Lsm1 to Lsm7 is marked as " Lsm1 - 7 " values correspond to mean of values found in purifications ; CK2 group corresponds to Cka1 , Cka2 , Ckb1 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 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 Edc3 - HA . 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 x - axis showing average enrichment value ( log2 scale ) for Upf1 - decapping 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 x - axis showing average enrichment value ( log2 scale ) for Upf1 - decapping Upf1 - 23 components . colour of bars illustrates treatment used during purification , black without RNase , orange with an RNase treatment . Error bars represent SD . dots correspond to enrichment values obtained in

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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 enrichment of Upf1 , Nmd4 Ebs1 in purification of Upf1 fragments . Upf1 C - terminal region ( 854 - 971 ) affected association with Nmd4 or Ebs1 ( Student t - with a one - sided alternative hypothesis ) . We compared enrichment of each of Ebs1 Nmd4 between purifications of Upf1 fragments having this region ( Upf1 - FL Upf1 - HD - Cter , 6 experiments ) purifications of Upf1 fragments lacking extension ( Upf1 - CH - HD Upf1 - HD , 4 experiments ) . 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 eluates of Upf1 domains purification in a Nmd4 - HA . band with # might corresponds to a dimer of Upf1 - CH , bands marked with a star correspond to residual signal with anti - HA antibodies ( Nmd4 ) . Fragments in eluate have a smaller because A part of tag was removed by digestion with TEV protease . G6PDH served as a loading control in 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 Upf1 - decapping components in purified Upf1 - TAP in presence ( black bars ) absence ( blue bars ) of NMD4 . Pink blue vertical lines highlight of Upf1 - 23 Upf1 - decapping complexes respectively . dots correspond to enrichment values obtained in replicate experiments , 6 for Upf1 - TAP 3 for 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 presence ( black bars ) absence ( blue bars ) of UPF1 . dots correspond to enrichment values obtained in replicate experiments , 4 for Nmd4 - TAP 2 for 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 ( overexpressed in E . coli purified ) . 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 fragment was tested by mixing purified Upf1 helicase domain with Nmd4 testing fraction of recovered on streptavidin beads after washes with 150 mM 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 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 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 transcripts that were most increased in 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 . 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 5 bin 3 4 were significant ( binomial , p < 10 - 24 ) . D . Same as in C , for ebs1 ∆ . Differences between distribution of transcripts in bin n bin n + 1 were significant ( binomial , 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)