( G ) Immunoblot analysis of EphA2 and IL - 1β in lung homogenates were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice on day 1 and day 3 of intranasal infection with reovirus ( 4 × 104 PFU per mouse ) , β - actin served as a loading control throughout .

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(G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.


Quantitative RT - PCR analysis of mRNA encoding various cytokines ( IL - 4 , IL - 5 , IL - 13 in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

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

Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13 in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


Quantitative RT - PCR analysis of mRNA encoding various cytokines IL - 33 ) in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . ( C ) ELISA analysis of IL - 33 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

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

Quantitative RT-PCR analysis of mRNA encoding various cytokines IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. (C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( D - G ) ELISA analysis of IL - 4 , IL - 13 , IL - 1β and IL - 18 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

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(D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( H ) Cell number and category of BALF samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

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(H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( I ) H & E staining and PAS staining of lung sections from wild - type ( WT ) and EphA2 - knockout ( KO ) mice sensitized with ova . Scale bars represent 200μm .

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(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.


( A ) Immunoblot analysis of proteins precipitated with anti - EphA2 or IgG ( control ) were from whole - cell lysates of AECs from wild - type mice stimulated with reovirus ( MOI = 5 ) for 3 h . Input , 10 % of the AECs lysate . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

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(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( C ) The indicated plasmids of HA - tagged full - length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged NLRP3 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc Antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

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(C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( D ) The indicated plasmids of HA - tagged full - length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged EphA2 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

(D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( E ) Colocalization of endogenous EphA2 and NLRP3 in AEC cells . AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis . EphA2 was stained with rabbit anti - EphA2 ( 1 : 500 ) , followed by Alexa Fluor 594 goat anti - rabbit secondary antibody ( red ) , while NLRP3 was stained with mouse anti - NLRP3 ( 1 : 500 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) served as the nuclei marker ( blue ) . Scale bars represent 20μm . The Pearson ' s correlation coefficient and the Mander ' s overlap coefficient in the upper red box are 0 . 5 , and 0 . 95 respectively , and the lower red box 0 . 78 , and 0 . 94 respectively .

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(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.


( A ) The indicated plasmids of Myc - tagged NLRP3 and GFP - tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - GFP and anti - Myc .

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(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.


( B ) Immunoblot analysis of p - Tyr of NLRP3 precipitated with anti - NLRP3 antibody in AECs without ( Ctrl ) or with LPS stimulation or reovirus ( Reo ) infection .

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(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.


( C ) The indicated plasmids of Myc - tagged wild - type NLRP3 or its mutants Y132F , Y164F , Y251F , Y570F and Y589F were respectively transfected into HEK293T cells along with GFP - tagged EphA2 , and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - HA antibody and anti - Myc antibody .

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

(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.


( D ) Immunoblot analysis of EphA2 , ASC , NLRP3 , caspase - 1 and IL - 1β in HEK293T cells were co - transfected with 200ng EphA2 , 40ng ASC , 40ng caspase - 1 and 200ng IL - 1β , vector or wild NLPR3 or Y132F as indicated . The position of protein markers ( shown in kDa ) is indicated on the right - hand side .

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(D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side.


( E ) Densitometry analysis * P < 0 . 05 ( unpaired t test ) . NS means no statistical difference . Shown is mean ± SD . Data represent three independent biological replicates .

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(E) Densitometry analysis *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.


( A - B ) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection ( MOI ) of 20 for 3h . AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control . ASC was stained with mouse anti - ASC ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


( C - D ) Confocal microscopy analysis of HEK293T cells transfected with flag - ASC and Myc - tagged vector or Myc - tagged NLRP3 or Myc - tagged NLRP3 Y132F . Cells were stained with anti - flag ( 1 : 200 ) , anti - Myc ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - rabbit secondary antibody ( green ) or Alexa Fluor 594 goat anti - mouse secondary antibody ( red ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


A - B . SH - SY5Y cells and primary cortical neurons were treated with 1 μM α - syn fibrils ( A ) or monomers ( B ) for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to PBS , unpaired t - test .

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A-B. SH-SY5Y cells and primary cortical neurons were treated with 1 μM α-syn fibrils (A) or monomers (B) for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to PBS, unpaired t-test.


C . Frontal cortices of A53T α - syn heterozygous transgenic mice and control mice at the age of 9 months ( old ) or 2 months ( young ) were lysed and Western blot was performed with the indicated antibodies ( n = 3 ) . * * * P < 0 . 001 compared to WT , unpaired t - test .

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C. Frontal cortices of A53T α-syn heterozygous transgenic mice and control mice at the age of 9 months (old) or 2 months (young) were lysed and Western blot was performed with the indicated antibodies (n=3). *** P < 0.001 compared to WT, unpaired t-test.


FcγRIIB KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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FcγRIIB KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 1 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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SHP-1 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 2 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

SHP-2 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells with 20 μM NSC87877 , 10 μM SSG and 1 μM PHPS - 1 were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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

SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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

SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


NT , c - src KD # 1 and # 2 SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells , one - way

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

NT, c-src KD #1 and #2 SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA


, D . primary cortical neurons were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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

, D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


, E . primary cortical neurons downregulating c - src were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells one - way ANOVA unpaired t -

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

, E. primary cortical neurons downregulating c-src were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells one-way ANOVA unpaired t-test


SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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

SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


G . FcγRIIB KD SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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

G. FcγRIIB KD SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


B . A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 , respectively , with 50 μM RA for 5 days .

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B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.


C . NT or c - src KD # 1 or # 2 / A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured with 50 μM RA for 5 days . The samples were observed under confocal microscopy , and the number of cells containing double fluorescence labeled puncta were analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to PBS or NT cells , one - way ANOVA .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.


A . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h .

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A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h.


B . Differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells were cultured 12 h . Levels of α - syn in culture media were then measured using ELISA . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to control , one - way ANOVA .

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

B. Differentiated NT, c-src KD #1 or c-src KD#2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured 12 h. Levels of α-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to control, one-way ANOVA.


D . SH - SY5Y cells in the lower chamber were cocultured with differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells for 12 h . The cells in the lower chamber were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to NT cells , one - way ANOVA .

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

D. SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1 or c-src KD #2/ A53T α-syn-EGFP OE SH-SY5Y cells for 12 h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to NT cells, one-way ANOVA.


E . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 in the presence of DMSO or 50 μM bafilomycin A1 ( BafA ) for 12 h .

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E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.


Differentiated NT and c - src KD / A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


G . NT and c - src KD primary neurons were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

G. NT and c-src KD primary neurons were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


H . SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h . The cells were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * P < 0 . 01 , one - way ANOVA .

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

H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.


SH - SY5Y cells in the presence of 5 μM saracatinib , 10 μM SKI - 1 were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA .

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

SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.


B . NT , c - src KD # 1 or c - src KD # 2 SH - SY5Y cells were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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. NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.


Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . B . Fixed brain tissues containing the frontal cortex and the striatum were lysed . Western blot was performed with indicated antibodies . Values were derived from three individual mice ( n = 3 ) .

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

Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. B. Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).


Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . C . Immunohistochemistry of pSer129 α - syn was performed . Arrow indicates pSer129 α - syn positive inclusion . Scale bar indicates 50 μm . D . Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions . Values were derived from eight individual mice ( n = 8 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS group , unpaired t - test . Data are expressed as mean ± SEM .

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

Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm. D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM.


A . Immunohistochemistry of GFAP was performed . GFAP intensities in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

A. Immunohistochemistry of GFAP was performed. GFAP intensities in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


B . Immunohistochemistry of IBA - 1 and MHC Class II were performed . Arrow indicates MHC class II positive cells . Numbers of MCH class II positive cells in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , one - way ANOVA .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunohistochemistry of IBA-1 and MHC Class II were performed. Arrow indicates MHC class II positive cells. Numbers of MCH class II positive cells in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, one-way ANOVA.


A . RNA - seq of whole blood from symptomatic ( n = 26 ; median 8 days PIO ) and asymptomatic ( n = 30 ; median 3 days post - hospital admission ) COVID - 19 patients at the acute phase of infection ( SARS - CoV - 2 PCR - positive ) was performed . Only samples with RNA integrity number > 6 were sent for sequencing and included in the analysis . Volcano plot indicating DEGs between asymptomatic and symptomatic COVID - 19 patients , with thresholds of FDR < 0 . 05 and | FC | > 2 . Numbers of over - expressed and under - expressed genes are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNA-seq of whole blood from symptomatic (n=26; median 8 days PIO) and asymptomatic (n=30; median 3 days post-hospital admission) COVID-19 patients at the acute phase of infection (SARS-CoV-2 PCR-positive) was performed. Only samples with RNA integrity number >6 were sent for sequencing and included in the analysis. Volcano plot indicating DEGs between asymptomatic and symptomatic COVID-19 patients, with thresholds of FDR < 0.05 and |FC| > 2. Numbers of over-expressed and under-expressed genes are indicated.


B . Network analysis of significant immune mediators between symptomatic and asymptomatic COVID - 19 patients . Interactive relationships between the cytokines or chemokines were determined by STRING ( Search Tool for the Retrieval of Interacting Genes / Proteins ) analysis , with a confidence threshold of 0 . 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Network analysis of significant immune mediators between symptomatic and asymptomatic COVID-19 patients. Interactive relationships between the cytokines or chemokines were determined by STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) analysis, with a confidence threshold of 0.5.


C . SARS - CoV - 2 specific CD4 + non T follicular helper ( TFH ) cells from symptomatic ( n = 5 ) and asymptomatic COVID - 19 ( n = 5 ) patients were characterized with flow cytometry based on the expression of IL - 17a , IFN - γ , TNF - ⍺ , IL - 4 and IL - 10 upon SARS - CoV - 2 peptide stimulation . Representative plot overlay of unstimulated ( black ) on peptide stimulated ( red ) was performed on asymptomatic patient . Data are presented as Mean ± SD * * P < 0 . 01 ( Mann - Whitney U test ) .

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C. SARS-CoV-2 specific CD4+ non T follicular helper (TFH) cells from symptomatic (n=5) and asymptomatic COVID-19 (n=5) patients were characterized with flow cytometry based on the expression of IL-17a, IFN-γ, TNF-⍺, IL-4 and IL-10 upon SARS-CoV-2 peptide stimulation. Representative plot overlay of unstimulated (black) on peptide stimulated (red) was performed on asymptomatic patient. Data are presented as Mean ± SD **P<0.01 (Mann-Whitney U test).


A , B . ( A ) IgG and ( B ) IgM responses were analysed by screening plasma samples of asymptomatic ( n = 39 , median study day 29 ) and symptomatic ( n = 57 , median study day 31 ) COVID - 19 patients . Data are presented as Mean ± SD * * * P < 0 . 001 ( Mann - Whitney U test ) .

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A, B. (A) IgG and (B) IgM responses were analysed by screening plasma samples of asymptomatic (n=39, median study day 29) and symptomatic (n=57, median study day 31) COVID-19 patients. Data are presented as Mean ± SD ***P<0.001 (Mann-Whitney U test).


A . Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic ( n = 16 ) and symptomatic ( n = 52 ) COVID - 19 patients and healthy controls ( n = 17 ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


B . Mass cytometry was performed on PBMCs obtained from acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) . Monocytes were characterized based on their CD14 and CD16 expression . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . C . Classical , intermediate and non - classical monocytes were further defined in acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) with inflammatory marker CD169 . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


D . Expression data of genes associated with inflammatory monocytes , activated neutrophils and pro - inflammatory cytokines were compared between acute asymptomatic ( n = 30 ) and asymptomatic ( n = 26 ) COVID - 19 patients and were represented by ratio with respect to expression in asymptomatic patients . Bar graphs show DEGs with FDR adjusted P value < 0 . 05 and fold change values are represented in the log2 scale .

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D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.


E . Pro - inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45 - plex microbead - based immunoassay . Immune mediator levels were compared between asymptomatic patients ( n = 48 ) and symptomatic patients stratified by COVID - 19 severity ( mild , n = 61 ; moderate , n = 43 ; severe , n = 68 ) . Immune mediator levels for healthy controls ( HC ) ( n = 23 ) are indicated by the black dotted line . Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification ( LOQ ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( One - way ANOVA with post hoc t test ) .

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E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).


A . Growth factors in the plasma of asymptomatic ( n = 48 ) , symptomatic patients ( n = 173 ) and healthy controls ( n = 23 ) were analyzed by Luminex . A ratio of VEGF - A / VEGF - D was calculated and used to perform receiver operating characteristics analysis . The area under the curve was shown . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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A. Growth factors in the plasma of asymptomatic (n=48), symptomatic patients (n=173) and healthy controls (n=23) were analyzed by Luminex. A ratio of VEGF-A/VEGF-D was calculated and used to perform receiver operating characteristics analysis. The area under the curve was shown. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


B . RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown . Genes with false discovery rate less than 0 . 05 ( * ) , 0 . 01 ( * * ) or 0 . 001 ( * * * ) are indicated .

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B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.


C . Cell counts of DCs , CD4 T cells , CD8 T cells and B cells that express specific chemokine markers between asymptomatic ( n = 19 ) , symptomatic patients ( n = 37 ) and healthy controls ( n = 10 ) are analyzed by CyTOF and shown . Data are presented as Mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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C. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


A . Pull - down assays of yTBP ( 61 - 240 ) with Kap95p , Kap114p and Kap121p . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap95p , Kap114p and Kap121p . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . B . Band intensities of Kap - βs from the SDS - PAGE gels in ( A ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

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A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


C - E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( C ) Kap114p , ( D ) Kap95p , and ( E ) Kap121p with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

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C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.


A , B . Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC . SEC ( Superdex 200 ) elution profiles of the ( A ) Kap114p  RanQ69L and ( B ) Kap114p  yTBP complexes . Peak fractions were analyzed by SDS - PAGE and stained with Coomassie blue . The void volume ( Vo ) of the peak fraction and absorbance ( a . u . ) at 280 nm for each complex is indicated .

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A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.


SV - AUC analysis of ( C ) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

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SV-AUC analysis of (C) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


SV - AUC ( D ) Kap114p and ( E ) the Kap114p  yTBP complex . The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

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SV-AUC (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


F . SE - AUC analysis of the Kap114p  yTBP complex . The average molecular mass determined by the Sedfit program was ~ 133 . 5 kDa . The lower panel shows the residual difference ( standard deviation ) between experimental and fitted values .

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F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.


Plots of the distance distribution function P ( r ) for the ( G ) Kap114p  RanQ69L complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  RanQ69L ( H ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

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Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


The ab initio envelopes determined by GASBOR for the ( I ) Kap114p  RanQ69L complexes . Models of the ( J ) Kap114p  RanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR .

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The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L complexes. Models of the (J) Kap114pRanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.


Plots of the distance distribution function P ( r ) for the ( K ) Kap114p  yTBP complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  yTBP ( L ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

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Plots of the distance distribution function P(r) for the (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


B . Pull - down assays of yTBP with Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . C . Band intensities of Kap - βs from the SDS - PAGE gels in ( B ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

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B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


D , E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( D ) Kap114p ( Δ347 - 371 ) and ( E ) Kap114p ( Δ899 - 956 ) with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

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D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.


F . Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type , KAP114 knockout strains , and KAP114 knockout strains rescued by KAP114 deletion mutants .

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F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.


G . His - tagged RanQ69L was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . H . Band intensities of Kap - βs from the SDS - PAGE gels in ( G ) were quantified and normalized by His - Ran intensity . Quantitative plots show percent Kap - βs binding on His - Ran .

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G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran.


I . GST pull - down assays of yTBP ( 61 - 240 ) with Kap114p or Kap114p ( Δ347 - 371 ) in the presence or absence of RanQ69L . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p and His - tagged RanQ69L at a molar ratio of 1 : 4 : 8 Kap114p : yTBP : RanQ69L . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . J . Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L . Band intensities of Kap114p and Kap114p ( Δ347 - 371 ) from the SDS - PAGE gels were used to determine the average fraction of bound protein .

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I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.


B . EMSA analysis of yTBP binding to DNA . We incubated 5 nM FAM - labeled oligonucleotide ( TGTATGTATATAAAAC ) with indicated concentrations of purified yTBP ( 0 . 125 to 8 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

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B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


C . Indicated concentrations ( 0 . 25 to 16 μM ) of purified recombinant Kap114p were incubated with yTBP ( 4 μM ) and FAM - labeled DNA ( 5 nM ) , followed by native PAGE analysis . Kap114p ( 16 μM ) was incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

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C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


D . Super - shift EMSA analysis of TFIIA binding to yTBP  DNA complex . We incubated 5 nM FAM - labeled DNA and yTBP ( 0 . 5 μM ) with indicated concentrations of purified TFIIA ( 0 . 08 to 5 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

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D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


E . Indicated concentrations ( 0 . 06 to 4 μM ) of purified Kap114p were added to the reaction containing 0 . 5 μM of TFIIA , yTBP and FAM - labeled DNA ( TGTATGTATATAAAAC ) , before conducting native PAGE analysis . TFIIA and Kap114p ( 4 μM ) were incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . Electrophoretic bands containing protein - DNA complex and free DNA are indicated .

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E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.


A . Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1 . 5 M NaCl .

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A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl.


B . The KAP114 knockout strain was transformed with vector control ( lane 2 ) and a plasmid containing wild type KAP114 , KAP114 ( Δ347 - 371 ) , or KAP114 ( Δ899 - 956 ) ( lanes 3 - 5 ) . Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1 . 5 M NaCl . Growth temperatures are indicated .

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B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated.


C . A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1 . 5 M NaCl . The Log2 ( fold - change ) values and the color scale are shown .

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C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown.


E , F . ( E ) qPCR analysis of gene expression in the KAP114 knockout strains ( green bars ) compared to wild type ( black bars ) under regular conditions . ( F ) qPCR analysis of gene expression in the KAP114 knockout strain ( green bars ) compared to wild type ( black bars ) grown in high salt conditions . qPCR fold - changes in mRNA expression are relative to control ( YPD media , black bars ) . Actin was used as the internal control .

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E, F. (E) qPCR analysis of gene expression in the KAP114 knockout strains (green bars) compared to wild type (black bars) under regular conditions. (F) qPCR analysis of gene expression in the KAP114 knockout strain (green bars) compared to wild type (black bars) grown in high salt conditions. qPCR fold-changes in mRNA expression are relative to control (YPD media, black bars). Actin was used as the internal control.


G . ChIP - qPCR assays showing yTBP - GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions ( light green bars ) compared to under regular conditions ( black bars ) . Chip - qPCR fold - enrichment is relative to control ( YPD media , black bars ) .

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

G. ChIP-qPCR assays showing yTBP-GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions (light green bars) compared to under regular conditions (black bars). Chip-qPCR fold-enrichment is relative to control (YPD media, black bars).


H . Endogenous Ran , GFP - yTBP and proliferating cell nuclear antigen ( PCNA ; loading control ) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran , GFP and PCNA . I , J . Analysis of Ran and GFP - yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions . The combined bar and scatter plot shows the band intensity ratios of Ran ( I ) and GFP - yTBP ( J ) over PCNA . Each dot represents an individual data point .

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

H. Endogenous Ran, GFP-yTBP and proliferating cell nuclear antigen (PCNA; loading control) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran, GFP and PCNA. I, J. Analysis of Ran and GFP-yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions. The combined bar and scatter plot shows the band intensity ratios of Ran (I) and GFP-yTBP (J) over PCNA. Each dot represents an individual data point.


SUMO conjugation assays performed under multiple turnover conditions using wild - type or mutant forms of RPA , SUMO and Siz2 . Only the 80 - minute time points are presented to highlight the formation of Rfa1 - SUMO and Siz2 - SUMO species . The complete time series are presented in Appendix Fig S1 . Bands for Mw marker are fully annotated in Figure 1B .

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 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

SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions using Siz2 ∆ CT and different RPA - DNA complexes . SDS - PAGE gels were stained with Coomassie . Bands for Mw marker are fully annotated in Figure 1B .

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

SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.


Plot showing the initial rate of SUMO conjugation versus RPA - DNA concentration as obtained from single - turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA - DNA complexes . Representative gels are shown in Appendix Fig S4 .

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

Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.


SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2 ∆ CT lacking ( A ) or including ( B ) its SAP domain . In both cases , reactions were done in technical triplicate and SDS - PAGE gels were stained with SYPRO . Only one representative gel is shown . Bands for Mw marker are fully annotated in Figure 1B .

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

SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 . Experiments were performed in technical triplicates ( six times for WT ) . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

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

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2 ∆ CT variants . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

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

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


Electrophoretic mobility shift assays ( EMSA ) to assess binding of RPA or SUMO - RPA to 25nM single - stranded 6 - FAM - labeled 32dT DNA and displacement of SUMO - RPA from 25nM SUMO - RPA bound to single - stranded 6 - FAM - labeled 32dT DNA by RPA and vice versa . All titrations were done in technical triplicates . One representative gel is shown .

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

Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.


Electrophoretic mobility shift assays ( EMSA ) performed with 10 nM 6 - FAM - ds20ss32 , a 6 - FAM - labeled 20 - mer dsDNA with a 32 nucleotide 3 ' overhang and its complexes with RPA , RPA with K170 - SUMO modified Rfa1 or RPA reconstituted with ∆ WH - Rfa2 and serially diluted Siz2 . All titrations were done in technical triplicates . One representative gel is shown .

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

Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.


C . Total USV number in YFO and AFO . Data information : Data are presented in the box plot or pie chart format . The central band in each box represents the median ; boxes indicate the middle quartiles , and whiskers extend to the minimum and maximum values . * p < 0 . 05 , determined using Student ' s or Welch t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total USV number in YFO and AFO. Data information: Data are presented in the box plot or pie chart format. The central band in each box represents the median; boxes indicate the middle quartiles, and whiskers extend to the minimum and maximum values. *p<0.05, determined using Student's or Welch t-test.


D . A heat map of the 16 hyper - and 96 hypo - methylated DMRs in sperm from aged mice ordered by their chromosomal positions . M indicates the average .

O O O O O O O O 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. A heat map of the 16 hyper- and 96 hypo-methylated DMRs in sperm from aged mice ordered by their chromosomal positions. M indicates the average.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by nominal GSEA 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by nominal GSEA p-value.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by volcano or nominal GSEA P - value . NES , normalized enrichment score .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by volcano or nominal GSEA P-value. NES, normalized enrichment score.


C . Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Satb2 + / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Satb2 - / Ctip2 - : layer 6 . Scale bar : 100 μm . F . Cell number in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

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

C. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm. F. Cell number in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


D . Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Tbr1 - / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Tbr1 + / Ctip2 - : layer 6 . Scale bar : 100 μm . G . Cell density in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

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

D. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm. G. Cell density in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


C . Representative image of 5 - mC immunofluorescence in the sperm of saline - and 5 - Aza - treated mice ( ST and AT , respectively ) . Scale bar : 100 μm . D . Quantification analysis of 5 - mC immunofluorescence in individual sperm cells from STO ( n = 1 , 673 sperm from 4 male mice ) and ATO ( n = 2 , 675 sperm from 4 male mice ) . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

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

C. Representative image of 5-mC immunofluorescence in the sperm of saline- and 5-Aza-treated mice (ST and AT, respectively). Scale bar: 100 μm. D. Quantification analysis of 5-mC immunofluorescence in individual sperm cells from STO (n=1,673 sperm from 4 male mice) and ATO (n=2,675 sperm from 4 male mice).Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


F . Overall USV number in STO and ATO . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

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

F. Overall USV number in STO and ATO. Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


( A ) Distribution of antimycin A resistance scores for wild isolates , compared to standard laboratory strain ( red vertical line ) . Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg / L antimycin A . Fitness was estimated based on colony size on solid media , corrected for spatial and plate effects After quality control , we obtained quantitative fitness scores for 154 strains , with a signal - to - noise ratio of 29 . 8 and an unexplained variance of 0 . 12 .

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

(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.


( B ) Volcano plot of a genome - wide association using mixed - model linear regression of antimycin A resistance for 118 , 527 genetic variants . Variants with moderate or high impact are shown in blue . Red dot : the variant at locus I : 3845516 , which causes a T343A change in the Pyk1 amino - acid sequence . This variant was among the top scoring ( effect size = - 0 . 645 , rank 34 ; p = 0 . 0008 , rank 70 ) .

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

(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).


( C ) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus . Strains carrying a C at this genomic locus ( orange box ) generally have higher antimycin resistance than strains carrying the reference allele T ( blue box ) . As is standard , this boxplot and all other boxplots in this manuscript show the median of the data as central line , the quartiles as box and the extend of the rest of the distribution as whiskers . Points which are 1 . 5 times the inter - quartile range beyond the high and low quartiles are considered outliers and shown individually .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.


( B ) Left boxplot : two glycolytic intermediates directly upstream of PYK were strongly depleted in the A - strain ( nT - strain = 9 , nA - strain = 8 ) . Right barplot : PYK activity was directly measured using a lactate dehydrogenase - coupled colorimetric enzyme activity assay , showing the mean and standard deviation of substrate conversion rate for 3 biological replicates , each measured in technical duplicates . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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

(B) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates. Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( C ) Concentrations of fructose - 1 , 6 - bisphosphate , which correlate with glycolytic flux , are significantly higher in A - strain ( ratio = 1 . 35 , nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( D ) Boxplots for selected metabolomics data indicate differences in energy and redox status between T - and A - strains ( nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( E ) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T - and A - strains ( FDR < 10 % ) and are measured in all four conditions ( columns ) . First column : genes ordered by increasing fold - changes for RNAs ( computed as log2 [ T ] - log2 [ A ] ) . Second column : fold - changes for proteins ( computed as log2 [ T ] - log2 [ A ] ) . Third column : fold - changes for RNAs in cells treated with rapamycin & caffeine ( TORC1 inhibition ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from ref . ( Rallis et al , 2013 ) ) . Fourth column : fold - changes for RNAs in cells treated with H2O2 ( oxidative stress ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from Chen et al , 2003 ) . Log2 fold - changes are capped at absolute values of 1 for all columns .

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

(E) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.