( G ) FRET efficiency for AXL - OPCML interaction in SKOV3 - Empty " E " SKOV3 - OPCML " O " , using AXL - AXL FRET as a positive control . were labeled with Anti - Axl rabbit ( conjugated to donor probe ) Anti - OPCML mouse ( conjugated to acceptor probe ) , or Anti - Axl mouse ( conjugated to acceptor probe ) Anti - Axl rabbit ( conjugated to donor probe ) Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , * * * * P < 0 . 0001 by Student " s t

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

(G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( H ) PLA of AXL - OPCML ( red ) in SKOV3 PEA1 transduced with empty vector " E " or OPCML " O " . Scale bar =

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

(H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μm


ovarian tumour were characterised for AXL OPCML by ( I ) Western blottin PAX8 was used to identify ovarian cancer

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

Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cells


ovarian tumour were characterised for AXL OPCML b ( J ) Immunostaining . PAX8 was used to identify ovarian cancer

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

Primary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells


( K ) Quantitation of AXL - OPCML PLA in Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± s ; * P < 0 . 05 , * * * P < 0 . 01 , * * * * P < 0 . 0001 by Student " s t

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

(K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( L ) Western blotting of fractionation of SKOV3 - Empty : total lysate " T " , liquid - disordered soluble fraction " S " , detergent resistant fraction " R " . Caveolin - 1 is used as a marker of R fraction Calnexin is used as a marker for S fraction , E : SKOV3 - Empty , O : SKOV3 -

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

(L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML


( M ) Densitometry of AXL band intensities in ( J ) in S R , normalized to AXL in input . Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , * * * * P < 0 . 0001 by Student " s t

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

(M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


SKOV3 - OPCML stimulated with Gas6 over a 12 hour time course ( A ) stained with DAPI ( cyan ) , Anti - OPCML ( green ) Anti - AXL ( red ) antibodies ( scale bar = 10μm )

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

SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm)


( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) quantified , n = 3 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


d ; ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) quantified , n =

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

d; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3


( E ) Quantitation OPCML - AXL PLA interaction in ovarian tumour , n = 1 Data i M , ( E ) was performed once due to limited numbers of ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( F ) Co - immunoprecipitation with anti - AXL antibody in stimulated with Gas6 as indicated . Input : 1 / 50 of whole lysate from SKOV3 - OPCML . IP : Immunoprecipitated , IB : Immunoblotted

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

(F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( G ) Co - immunoprecipitation with anti - pAXL antibody in SKOV3 - OPCML stimulated with

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

(G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6


( H ) OPCML - GST pull down in SKOV3 - Empty stimulated with

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

(H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6


( I ) fractionation in SKOV3 - Empty " E " SKOV3 - OPCML " O " into detergent resistant " R " fraction detergent soluble " S " fraction upon Gas6

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

(I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( K ) FRAP recovery curves in insoluble lipid domain or soluble plasma fraction in SKOV3 - Empty " E " SKOV3 - OPCML " O " treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( L ) Diffusion Co - efficient in insoluble lipid domain or soluble plasma fraction in SKOV3 - Empty " E " SKOV3 - OPCML " O " treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( M ) of AXL localised in insoluble lipid domain or soluble plasma fraction in SKOV3 - Empty " E " SKOV3 - OPCML " O " treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( N ) FRET Efficiency in SKOV3 - OPCML " O " stimulated with Gas6 , labeled with Anti - Axl ( donor probe ) Anti - OPCML ( acceptor probe ) Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student " s t

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

(N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( O ) Western blotting of " S " " R " fractions in SKOV3 - Empty " E " OCPML " O " stimulated with Gas6 for 3 hours , for Gas6 , AXL pAXL . Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ±

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-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) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM


( A ) Filipin of SKOV3 - OPCML treated for 30 minutes with 5mM MBCD allowed to recover for 0 , 30 minutes , 12 hours in serum free ( SF )

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

(A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium


( B ) Western blotting of SKOV3 - OPCML treated with MBCD separated into detergent resistant " R " fraction detergent soluble " S " fraction upon Gas6

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

(B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( C ) of AXL band in " R " or pAXL band in " S " relative to total AXL " T " from SKOV3 - OPCML " O " in ( B )

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

(C) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B)


( D ) MBCD treatment of SKOV3 - EMPTY SKOV3 - OPCML lines was followed by of Gas6 signalling kinetics by western blot . GAPDH was used as loading

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

(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control


( E , F ) Signalling kinetics are presented for pAXL pERK in SKOV3 - EMPTY " E " SKOV3 - OCPML " O " , treated with MBCD no treatment control , normalised to GAPDH , from ( D )

O O O O 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

(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)


( G ) PLA of AXL - OPCML interaction ( red ) upon Gas6 stimulation in both MBCD treated non - treated SKOV3 - OPCML . Scale bar =

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

(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm


( B ) Western Blotting of SKOV3 - Empty " E " SKOV3 - OPCML " O " pre incubated with either no drug ( DMSO only control ) , 1 . 09 μM R428 , or 2 . 84 μM R428 for an hour stimulated with Gas6 for indicated times . GAPDH was used as loading

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

(B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control


( C ) Immunofluorescence of pERK ( red ) OPCML ( magenta ) in SKOV3 - Empty " E " SKOV3 - OPCML " O " treated with R428 stimulated with Gas6 for indicted time points . Scale bars =

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

(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm


D Western blot of Sec63 , mCherry Pgk1 from WT ( SSY1404 ) , harboring estradiol - inducible system ( SSY1405 ) or ∆ opi1 ( SSY1607 ) , of which expressed Sec63 - mNeon Rtn1 - mCherry . were untreated or treated with 800 nM estradiol for 6 h . Pgk1 served as a loading control .

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

D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.


E of ER in estradiol - treated harboring inducible system ( SSY1405 ) , untreated ∆ opi1 ( SSY1607 ) WT ( SSY1404 ) treated with 8 mM DTT for 1 h . Bars represent mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with 0 nM estradiol or ∆ opi1 , as judged by a two - Student ' s t - assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 ; n . s . , not significant .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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 Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.


A Sec63 - mNeon of of indicated genotypes harboring inducible system . were treated with 800 nM estradiol for 6 h . Two examples of each phenotypic class are shown .

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

A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.


C Sec63 - mNeon Rtn1 - mCherry of ∆ lnp1 ∆ sey1 harboring inducible system treated with 800 nM estradiol for 6 h .

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

C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.


C of peripheral ER structures in WT ∆ ice2 harboring inducible system ( SSY1405 , 1603 ) treated with 800 nM estradiol for times indicated . Bars are mean of cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for sum of tubules sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with corresponding value in WT , as judged by a two - Student ' s t - assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

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

C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


E of peripheral ER structures in untreated WT , Δice2 , Δopi1 Δice2 Δopi1 ( SSY1404 , 2356 , 2595 , 2811 ) . Bars are mean of cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for sum of tubules sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with corresponding value in WT , as judged by a two - Student ' s t - assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


C , D of cortical sections of WT Δice2 expressing Sec63 - mNeon Rtn1 - mCherry ( SSY1405 , 1603 ) that were untreated ( C ) or treated with 8 mM DTT for 1 h ( D ) .

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 B-CONTROLLED_VAR O O O O O O O

C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).


B Growth assays of untreated WT , Δice2 , Δnem1 , Δnem1 Δice2 , Δspo7 , Δspo7 Δice2 , Δpah1 Δpah1 Δice2 ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 , 2807 , 2808 ) . Numbers represent areas under curves serve as growth indices . Mean + s . e . m . , n = 3 biological replicates . Data for WT ∆ ice2 are same in left middle panels .

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

B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.


C , D Lipidomic of WT , Δice2 , Δnem1 , Δice2 Δnem1 , Δspo7 Δice2 Δspo7 ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT , as judged by a two - Student ' s t - assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 . Data for WT Δice2 are same as in both panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.


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

E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).


D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1 - HA from ∆ nem1 mutants ( SSY3065 ) microsomes from of indicated genotypes ( SSY3053 , 3074 , 3075 , 3095 ) . Phosphorylated Pah1 dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase ( PPase ) are shown for reference .

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

D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.


E Western blot of HA , Sec61 Pgk1 from lysates microsomes prepared from WT ∆ ice2 expressing Nem1 - HA ( SSY3140 , 3141 ) . Nem1 is undetectable in lysates due to its low abundance .

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

E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.


Western blots of FLAG , HA Dpm1 from lysates or anti - FLAG immunoprecipitates of WT or ∆ nem1 containing Spo7 - FLAG , Nem1 - FLAG or Ice2 - HA as indicated ( SSY122 , 2421 , 3183 , 3184 , 3195 , 3196 , 3197 ) . asterisk marks position of chain from antibody used for immunoprecipitation . diamond marks a that is non - specifically precipitated by anti - FLAG antibody . IP , immunoprecipitate .

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

Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.


E of with endogenously tagged Ice2 - mScarlet Spo7 - mNeon or Nem1 - mNeon ( SSY3244 , 3245 ) . Arrows indicate foci containg both Ice2 either Spo7 or Nem1 . F of Spo7 Nem1 foci in WT ∆ ice2 ( SSY2916 , 3238 , 2917 , 3239 ) . Mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with respective WT , as judged by a two - Student ' s t - assuming equal variance . * * , P < 0 . 01 .

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

E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1. F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


A Lipidomic of WT ∆ ice2 in which PAH1 was replaced with PAH1 - HA or pah1 ( 7A ) - HA as indicated ( SSY2841 , SSY2842 , SSY2970 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT , as judged by a two - Student ' s t - assuming equal variance . * * , P < 0 . 01 .

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

A Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


A Sec63 - mNeon of mid cortical sections of untreated WT ∆ opi1 , overexpressing ICE2 where indicated ( SSY1404 , 2588 , 2595 , 2596 ) .

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

A Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).


D Growth assays on solid media of WT , ∆ hac1 , Δice2 , Δhac1 Δice2 ( SSY1404 , 2356 , 2805 , 2806 ) in absence or presence of 0 . 2 µg / ml tunicamycin . For each series , were diluted fivefold from one step to next .

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-CONTROLLED_VAR O O O O O O O O O O O O O O

D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.


( A - F ) Immunocytochemistry of DRG neurons corresponding quantifications of indicated genotypes for surface TrkA ( A ) , surface TRPV1 ( B ) , Rab3A ( C ) , synaptotagmin ( D ) , DENN / MADD ( E ) α - tubulin ( F ) . TrkA TRPV1 were visualized without permeabilization . ns , p > 0 . 05 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t from at least three independent cultures . Error bars show . Scale bars , 2 µm .

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

(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm.


Time - lapse of DRG neurons expressing KIF1A - EGFP KIF1A ( E239K ) - EGFP ( B ) Arrowheads in ( B ) show anterogradely moving particles . Scale bar in ( B ) , 2 μm . See Movies EV1 2 .

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

Time-lapse fluorescence images of DRG neurons expressing KIF1A-EGFP and KIF1A(E239K)-EGFP (B) Arrowheads in (B) show the anterogradely moving particles. Scale bar in (B), 2 μm. See Movies EV1 and 2.


( F ) Movements of KIF1A - EGFP KIF1A ( E239K ) - EGFP expressed in KIF1A knockdown DRG scrambled control . scale bar , 1 μm . ( G ) Anterograde velocities of indicated genotypes calculated from kymograph slopes . Data are shown as mean SD . KD + KIF1A - EGFP , n = 29 ; KD + KIF1A ( E239K ) - EGFP , n = 32 ; scrambled + KIF1A - EGFP , n = 44 ; scrambled + KIF1A ( E239K ) - EGFP , n = 42 from three independent cultures . ns , p = 0 . 3329 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Unpaired t .

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

(F) Movements of KIF1A-EGFP and KIF1A(E239K)-EGFP expressed in KIF1A knockdown DRG and the scrambled control. White scale bar, 1 μm. (G) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes. Data are shown as mean and SD. KD+KIF1A-EGFP, n = 29; KD+KIF1A(E239K)-EGFP, n = 32; scrambled+KIF1A-EGFP, n = 44; scrambled+KIF1A(E239K)-EGFP, n = 42 from three independent cultures. ns, p = 0.3329; **, p < 0.01; ****, p < 0.0001; Unpaired t test.


( A ) Representative kymographs of tetramethylrhodamine - labelled microtubule movement driven by WT E239K mutant . arrowheads represent end positions of WT E239K .

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

(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.


( D ) ATPase activity of WT ( green ) E239K mutant ( magenta ) activated by microtubules . Data are represented as mean ± SD . data from at least three independent experiments were analysed . * * * * , p < 0 . 0001 , two - way ANOVA .

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

(D) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA.


( F ) Rate constants ( kobs ) determined for reaction of mant - ATP with nucleotide - free KIF1A ( WT ) KIF1A ( E239K ) were plotted against of mant - ATP . Data are represented as mean ± SD . data from three independent measurements were analysed .

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-MEASURED_VAR O O O B-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

(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.


( A ) positions of β6 - L10 - β7 neck - coil peptides are shown in brown green . positions of neck - linker peripheral peptides ( α1 - β3 - P loop , α4 - L12 L12 - α5 ) are shown in blue . ( B - D ) Quantitation of site - specific intramolecular interactions . Representative MS / MS spectra of indicated peptides , including mutated acid ( β6 - L10 - β7 ) ( B C ) neck - coil ( D ) . In inserted graphs , intensities in ADP - BeFx ADP - AlFx states were measured with respect to nucleotide - free state as 1 . 0 . Grey bar graphs , WT ; black bar graphs , E239K ; ( - ) , nucleotide - free .

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

(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free.


( e ) Binding of monoclonal antibodies to 2019 - nCoV RBD determined by ELISA .

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

(e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA.


( f ) Binding profiles of 2019 - nCoV RBD to ACE2

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

(f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies


( g ) competition of CR3022 ACE2 with 2019 - nCoV RBD measured by BLI in OctetRED96 . Binding kinetics was evaluated using a 1 : 1 Langmuir binding model by ForteBio Data 7 . 0 software .

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

(g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.


C Comparison of meiotic chromosome segregation in lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary IF microscopy of indicated fission yeast strains are shown . Homothallic strains were incubated on SPAS plates . tubulin were stained with DAPI anti - TAT - 1 , respectively . Missegregating chromosome is indicated with a yellow arrow ; D of meiotic chromosome segregation . data for clr4 + clr4F449Y is same as in Fig 3B . P values were calculated using Fisher ' s exact . Number of total counted is indicated in each bar .

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

C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.


E Comparison of co - segregation of centromere I ( CenI ) during MI in lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary live microscopy of indicated fission yeast strains during MI MII are shown . Heterothallic strains were crossed dyads as well as tetrads were subjected to . Heterozygous CenI - GFP was used to follow segregation pattern during meiosis . GFP - signal is additionally displayed in greyscale for better visibility F of CenI - GFP segregation during meiosis . data for clr4 + clr4F449Y is same as in Fig 3D . P values were calculated using Fisher ' s exact . Number of total counted is indicated in each bar .

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 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

E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.


G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1 - like - CD . Exemplary image ( left ) of spore viability ( right ) are shown . Meiosis was induced in homothallic resulting spores were dissected . data for clr4 + clr4F449Y is same as in Fig 3F . P values were calculated using Fisher ' s exact . Number of dissected tetrads is indicated in each bar .

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 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

G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1-like-CD cells. Exemplary image (left) and quantification of the spore viability (right) are shown. Meiosis was induced in homothallic cells and the resulting spores were dissected. The data for clr4+ and clr4F449Y is the same as in Fig 3F. P values were calculated using Fisher's exact test. Number of dissected tetrads is indicated in each bar.


A Western blot of whole - lysates ( WCL ) using a PhosTag gel for anti - FLAG ( top ) a Bis - Tris gel for anti - Act1 detection ( bottom ) , respectively . WCL were prepared from mitotic meiotic 3xFLAG - clr4 + / 3xFLAG - clr4 + cultures at indicated time points .

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

A Western blot of whole-cell lysates (WCL) using a PhosTag gel for anti-FLAG (top) and a Bis-Tris gel for anti-Act1 detection (bottom), respectively. WCL were prepared from mitotic and meiotic 3xFLAG-clr4+/3xFLAG-clr4+ cultures at the indicated time points.


C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation of Clr4 in indicated homothallic strains .

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

C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains.


D Western blot of FLAG IPs in 3xFLAG - clr4 + cdc2 - as , treated with 2 µM 1 - NM - PP1 for indicated amount of time . blot was first probed with phospho - specific anti - pS458 antibody , stripped , reprobed with an anti - FLAG antibody .

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

D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cultures mixed with PBS OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants fractions were collected at indicated time points post - inoculation analyzed for : Infectivity in culture supernatants by plaque titration Data information : Data were generated in four experiments using from at least four donors represented by different symbols , bars represent mean , error bars indicate S . E . M . Statistical significance was tested using paired Student ' s t - comparing mock - Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant are not shown in figure . n . d . = not detectable ; h . p . e . = hours post - exposure Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.


PBMCs from four donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cultures mixed with PBS OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants fractions were collected at indicated time points post - inoculation analyzed for : Infectivity in culture supernatants by plaque titration Data information : Data were generated in four experiments using from at least four donors represented by different symbols , bars represent mean , error bars indicate S . E . M . Statistical significance was tested using paired Student ' s t - p - values > 0 . 05 were considered not significant are not shown in figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

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

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cultures mixed with PBS OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants fractions were collected at indicated time points post - inoculation analyzed for : Viral ( genome equivalents / ml ) concentrations in culture supernatants by Q - RT - PCR Data information : Data were generated in four experiments using from at least four donors represented by different symbols , bars represent mean , error bars indicate S . E . M . Statistical significance was tested using paired Student ' s t - comparing mock - Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant are not shown in figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; Ruxo. = Ruxolitinib.


PBMCs from four donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cultures mixed with PBS OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants fractions were collected at indicated time points post - inoculation analyzed for : Viral ( genome equivalents / ml ) concentrations in culture supernatants by Q - RT - PCR Data information : Data were generated in four experiments using from at least four donors represented by different symbols , bars represent mean , error bars indicate S . E . M . Statistical significance was tested using paired Student ' s t - p - values > 0 . 05 were considered not significant are not shown in figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

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

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cultures mixed with PBS OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants fractions were collected at indicated time points post - inoculation analyzed for : ( E ) Relative changes of - associated viral genomic quantities by Q - RT - PCR normalized to RNASEP . Data information : Data were generated in four experiments using from at least four donors represented by different symbols , bars represent mean , error bars indicate S . E . M . Statistical significance was tested using paired Student ' s t - comparing mock - Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant are not shown in figure . h . p . e . = hours post - exposure Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: (E) Relative changes of cell-associated viral genomic RNA quantities by Q-RT-PCR and normalized to RNASEP levels. Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.


extracted from Ruxolitinib - treated or mock - treated , SARS - CoV - , SARS - CoV - 2 - PBMCs was analyzed for ( A ) IFIT1 , ( B ) IFNA1 IFNB1 mRNA expression by Q - RT - PCR at indicated time points . Suspension adherent fractions were analyzed separately , except at four hours time point . Values were normalized to RNASEP expression are shown as fold change over mock - inoculated conditions . dotted indicates expression level of mock - inoculated cultures is set to 1 . Data information : Data were generated in four experiments using PBMCs from four or more donors represented by different symbols , bars represent mean , error bars indicate S . E . M ( A - B Statistical significance between mock - Ruxolitinib - treated samples was tested using paired Student ' s t - testing comparing SARS - CoV to SARS - CoV - 2 - treated samples from same donor time points . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) .

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

RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- PBMCs was analyzed for (A) IFIT1, (B) IFNA1 and IFNB1 mRNA expression by Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours time point. Values were normalized to cellular RNASEP expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( C ) Supernatants from Ruxolitinib - or mock - treated SARS - CoV - , SARS - CoV - 2 or mock - inoculated PBMCs were collected 48 hours post exposure cytokine expression of IFN - α2 , IP - 10 / CXCL10 , MCP - 1 / CCL2 MCP - 3 / CCL7 were quantified using a Luminex - immunoassay . PHA - or LPS - treated PBMCs were used as a positive control . Bars represent results of a pool of four samples per condition . Data information : Data were generated in four experiments using PBMCs from four or more donors represented by different symbols , bars represent mean , Statistical significance between mock - Ruxolitinib - treated samples was tested using paired Student ' s t - testing comparing SARS - CoV to SARS - CoV - 2 - treated samples from same donor P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) . n . d . = not detectable .

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

(C) Supernatants from Ruxolitinib- or mock-treated and SARS-CoV-, SARS-CoV-2 or mock-inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2 and MCP-3/CCL7 were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs were used as a positive control. Bars represent the results of a pool of four individual samples per condition. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.


( D ) Supernatants collected from SARS - CoV - , SARS - CoV - 2 - or mock - exposed PBMCs at indicated time points were analyzed for release of bioactive IFN using HL116 reporter . Data information : Data were generated in four experiments using PBMCs from four or more donors represented by different symbols , bars represent mean , error bars indicate S . E . M P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not 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 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

(D) Supernatants collected from SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( A ) Pseudotime trajectory GSEA using genes differentially regulated between mock - , SARS - CoV - SARS - CoV - 2 - challenged conditions for indicated types . Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O O O O 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) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock-, SARS-CoV- and SARS-CoV-2-challenged conditions for indicated cell types. Data information: Data shown in this figure are based on the analysis of two donors.


( B ) Representative UMAPs showing IFIT1 ISG15 mRNA expression in indicated conditions . Data information : Data shown in this figure are on of two donors .

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

(B) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions. Data information: Data shown in this figure are based on the analysis of two donors.


( C ) Volcano plot of DEGs in SARS - CoV - 2 - exposed compared to mock - exposed in indicated types . Known ISGs were colored in green on their presence in interferome database Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot of all DEGs in SARS-CoV-2-exposed cells compared to mock-exposed cells in the indicated cell types. Known ISGs were colored in green based on their presence in the interferome database Data information: Data shown in this figure are based on the analysis of two donors.


( D ) trajectory maps of indicated types with colored by expression of genes in an IFN - module gene set . Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O 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) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN-module gene set. Data information: Data shown in this figure are based on the analysis of two donors.


( E ) Dot plot depicting expression of selected ISGs cytokines . Expression are color - coded , of expressing respective gene is coded by symbol . Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O 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) Dot plot depicting expression of selected ISGs and cytokines. Expression levels are color-coded, the percentage of cells expressing the respective gene is coded by symbol size. Data information: Data shown in this figure are based on the analysis of two donors.


( C ) Bar graph showing absolute number ( bars ) relative of that were identified as - Positive in SARS - CoV - SARS - CoV - 2 - inoculated cultures , respectively . Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph showing the absolute number (bars) and relative percentage of cells that were identified as virus RNA-Positive in SARS-CoV- and SARS-CoV-2-inoculated cultures, respectively. Data information: Data shown in this figure are based on the analysis of two donors.


( D ) Plot of Log10 average expression of genes showing a Log2 ( fold change ) > 0 . 2 in viral - Positive versus viral - Negative monocytes from SARS - CoV - ( left panel ) SARS - CoV - 2 - ( right panel ) inoculated PBMCs with genes showing highest expression fold change between both conditions . Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plot of Log10 average expression of genes showing a Log2(fold change) >0.2 in viral RNA-Positive versus viral RNA-Negative monocytes from the SARS-CoV- (left panel) and SARS-CoV-2- (right panel) inoculated PBMCs with genes showing the highest expression fold change between both conditions. Data information: Data shown in this figure are based on the analysis of two donors.


( E ) IFN Module Score of viral - - negative ( grey ; 715 958 in SARS - CoV SARS - CoV - 2 exposed cultures , respectively ) , SARS - CoV - - Positive ( blue ; 56 ) SARS - CoV - 2 - - Positive ( magenta ; 173 ) monocytes . Statistical significance was tested using a Wilcoxon rank sum with continuity correction . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant Data information : Data shown in this figure are on of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IFN Module Score of viral-RNA-negative (grey; 715 and 958 cells in SARS-CoV and SARS-CoV-2 exposed cultures, respectively), SARS-CoV-RNA-Positive (blue; 56 cells) and SARS-CoV-2-RNA-Positive (magenta; 173 cells) monocytes. Statistical significance was tested using a Wilcoxon rank sum test with continuity correction. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant Data information: Data shown in this figure are based on the analysis of two donors.


Results of ELISAs used to determine IFN - β ( A ) secretion in PMA - differentiated THP - 1 transfected with siCtrl , siRNF34 - 1 , siRNF34 - 2 or siRNF34 - 3 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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 O O O

Results of the ELISAs used to determine IFN-β (A) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Results of ELISAs used to determine IL6 ( B ) secretion in PMA - differentiated THP - 1 transfected with siCtrl , siRNF34 - 1 , siRNF34 - 2 or siRNF34 - 3 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Results of the ELISAs used to determine IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Luciferase activity driven by IFN - β ( C ) promoter in HEK293T transfected with Flag - RNF34 or its E3 ligase - dead mutant H342A followed by VSV infection for indicated times . Luciferase assays were performed 24 hrs after transfection , luciferase activity was reported as fold induction . luciferase reporter data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Luciferase activity driven by the IFN-β (C) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Luciferase activity driven by NF - κB ( D ) promoter in HEK293T transfected with Flag - RNF34 or its E3 ligase - dead mutant H342A followed by VSV infection for indicated times . Luciferase assays were performed 24 hrs after transfection , luciferase activity was reported as fold induction . luciferase reporter data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

Luciferase activity driven by the NF-κB (D) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( E ) Plaque of VSV loads in supernatants from transfected with shCtrl , shRNF34 - 1 , shRNF34 - 2 or shRNF34 - 3 subsequently infected with VSV . - studies were performed independently at least three times with comparable results .

O O O O O O O O O O O O O O O O O O O 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) Plaque assay of VSV loads in supernatants from cells transfected with shCtrl, shRNF34-1, shRNF34-2 or shRNF34-3 and subsequently infected with VSV. Cell-based studies were performed independently at least three times with comparable results.


( F ) Microscopy of shCtrl - , shRNF34 - 1 - , shRNF34 - 2 - or shRNF34 - 3 infected with NDV - GFP ( MOI = 1 . 0 ) for indicated times . Gray represents ; green represents NDV . Scale bar , 300 μm .

(F) Microscopy images of shCtrl-, shRNF34-1-, shRNF34-2- or shRNF34-3 cells infected with NDV-GFP (MOI = 1.0) for the indicated times. Gray represents cells; green represents NDV. Scale bar, 300 μm.


( G ) Immunoblot showing of phosphorylated ( p ) total IRF3 , TBK1 , VSV - G in PMA - differentiated THP - 1 transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times .

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

(G) Immunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.


RT - PCR of expression of IFN - β ( H ) mRNAs in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . results were normalized to values obtained prior to VSV infection .

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

RT-PCR analysis of the expression of the IFN-β (H) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.


( I ) Results of ELISA used to determine IFN - β secretion in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

(I) Results of the ELISA used to determine IFN-β secretion in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


RT - PCR of expression of IL8 , ISG54 ISG56 ( J ) mRNAs in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . results were normalized to values obtained prior to VSV infection .

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

RT-PCR analysis of the expression of the IL8, ISG54 and ISG56 (J) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.


( A ) Luciferase activity driven by ISRE promoter in HEK293T transfected with Myc - RNF34 Flag - V , Flag - N - RIG - I , Flag - MAVS , Flag - STING or Flag - TBK1 . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

(A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( B ) Y2H in AH109 yeast co - transformed with indicated plasmids . A positive RNF34 - MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan , leucine , adenine , histidine containing X - gal . pGBKT7 - TP53 + pGADT7 - T pGBKT7 - lam + pGADT7 - T were used as positive negative controls , respectively . AH109 co - transfected with pGBKT7 - RNF34 + pACT - 2 was used to exclude self - activation of RNF34 .

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

(B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34.


( C ) Immunoprecipitation of HEK293T transfected with Myc - RNF34 Flag - MAVS or Flag - V . Anti - Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody .

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

(C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.


( D ) GST - tagged RNF34 was subjected to a pull - down with HEK293T lysates . Immunoblot with an anti - MAVS antibody are shown in top panel . Loading of GST assessed using Coomassie blue is shown in bottom panel . GST was used as a negative control .

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 O O O O 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) GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody are shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control.


( E ) Anti - Flag or IgG immunoprecipitates prepared from transfected with Flag - MAVS or Flag - vector - expressing plasmids were subjected to SDS - PAGE blotted onto a nitrocellulose . nitrocellulose was incubated with soluble GST - RNF34 ( upper panel ) or GST ( middle panel ) for 2 hrs then analyzed with anti - Flag antibody .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(E) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 hrs and then analyzed with anti-Flag antibody.


( F ) Representative confocal of immunofluorescence for Flag - RNF34 colocalization with endogenous MAVS in THP - 1 infected with VSV for 12 hrs . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

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

(F) Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 hrs. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoblot showing of RNF34 in THP - 1 infected with VSV ( MOI = 1 . 0 ) for indicated times . α - Tubulin was used as a loading control .

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

(G) Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control.


( H ) In situ PLA of RNF34 - MAVS in HEK293T infected with VSV ( MOI = 1 . 0 ) for indicated times using an anti - RNF34 or anti - MAVS antibody . RNF34 - MAVS , red ; nuclei , blue . Scale bar , 5 μm . - studies were performed independently at least three times with comparable results .

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

(H) In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI =1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 μm. Cell-based studies were performed independently at least three times with comparable results.


( I ) One hundred were counted , of PLA signals per is shown . - studies were performed independently at least three times with comparable results .

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

(I) One hundred cells were counted, and the quantification of PLA signals per cell is shown. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoprecipitation of HEK293T transfected with Myc - RNF34 Flag - MAVS or Flag - mMAVS . Anti - Flag immunoprecipitates were analyzed using immunoblotting with anti - Myc or anti - Flag antibody .

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

(J) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.


( A ) Immunoprecipitation in HEK293T expressing Flag - MAVS Myc - Ub together with HA - RNF34 or HA - H342A . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

(A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. The levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoprecipitation of HEK293T expressing Flag - MAVS HA - RNF34 together with Myc - Ub ( K6 , K11 , K27 , K29 , K33 , K48 , or K63 only ) as indicated . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

(B) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoprecipitation of HEK293T expressing Flag - MAVS HA - RNF34 together with Myc - Ub ( wild - type , WT ; Lys - to - Arg mutants of Ub lysines , KO ; K27 / 29R ) . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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 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

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( D ) Immunoprecipitation of K27 ubiquitination of MAVS in HEK293T infected with VSV for indicated times . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - Ub - K27 antibody . WCL was immunoblotted with an anti - MAVS or anti - Ub - K27 antibody . - studies were performed independently at least three times with comparable results .

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 B-MEASURED_VAR O O O B-MEASURED_VAR 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

(D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.


( E ) Immunoprecipitation of siCtrl - or siRNF34 - 1 - transfected infected with VSV for indicated times . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - Ub - K27 antibody . WCL was immunoblotted with an anti - MAVS or anti - Ub - K27 antibody . - studies were performed independently at least three times with comparable results .

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 B-MEASURED_VAR O O O B-MEASURED_VAR 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

(E) Immunoprecipitation analysis of siCtrl- or siRNF34-1-transfected cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.


Immunoprecipitation of HEK293T expressing Flag - MAVS or Lys to Arg mutants together with Myc - Ub HA - RNF34 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


Immunoprecipitation of HEK293T expressing Flag - MAVS or 4KR mutant together with Myc - Ub HA - RNF34 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Ub - K27 antibody . of transfected were analyzed by performing immunoblotting with an anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or 4KR mutant together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed by performing immunoblotting with an anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoprecipitation of HEK293T expressing Flag - MAVS or K311R mutant together with HA - RNF34 / or Myc - TRIM31 following a 3MA ( 0 . 2 mM ) treatment . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Flag , anti - Ub - K63 or anti - Ub - K27 antibody . of transfected were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . ell - studies were performed independently at least three times with comparable results .

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

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. ell-based studies were performed independently at least three times with comparable results.


( D ) Co - immunoprecipitation of ubiquitination of endogenous MAVS in THP - 1 transfected with siCtrl , siRNF34 , or siTRIM31 oligos infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

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

(D) Co-immunoprecipitation analysis of the ubiquitination of endogenous MAVS in THP-1 cells transfected with siCtrl, siRNF34, or siTRIM31 oligos and infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


( E ) Co - immunoprecipitation of endogenous interaction of MAVS - TRIM31 MAVS - RNF34 in PBMCs infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

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

(E) Co-immunoprecipitation analysis of the endogenous interaction of MAVS-TRIM31 and MAVS-RNF34 in PBMCs infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing degradation of MAVS in HEK293T subjected to VSV infection ( MOI = 1 . 0 ) for indicated times . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing degradation of MAVS in HEK293T infected with VSV ( MOI = 1 . 0 ) for indicated times in presence or absence of NH4Cl ( 3 mM ) . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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 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

(B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS or Flag - mMAVS together with Myc - RNF34 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or Flag-mMAVS together with Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( D ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS or its 4KR mutant together with increasing amounts of Myc - RNF34 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(D) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or its 4KR mutant together with increasing amounts of Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( E ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS with increasing amounts of HA - RNF34 or its H342A mutant . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( F ) SDD - of MAVS in HEK293T expressing Flag - MAVS together with WT Myc - RNF34 or its ligase - dead mutant cultured in presence or absence of MG132 ( 10 μM ) , 3 - MA ( 1 mM ) or NH4Cl ( 3 mM ) . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( G ) SDD - of MAVS in siCtrl - or siRNF34 - 1 - transfected using an anti - MAVS antibody . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O O O O B-MEASURED_VAR 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

(G) SDD-AGE analysis of MAVS aggregates in siCtrl- or siRNF34-1-transfected cells using an anti-MAVS antibody. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoblot showing of MAVS in siCtrl - or siRNF34 - 1 - transfected PBMCs . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(H) Immunoblot showing the levels of the MAVS protein in siCtrl- or siRNF34-1-transfected PBMCs. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( I ) SDD - of MAVS in THP - 1 expressing Flag - MAVS or K311R mutant together with Myc - RNF34 or Myc - TRIM31 . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(I) SDD-AGE analysis of MAVS aggregates in THP-1 cells expressing Flag-MAVS or the K311R mutant together with Myc-RNF34 or Myc-TRIM31. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoprecipitation of HEK293T transfected with Flag - SQSTM1 or its I431A mutant together with HA - MAVS . Anti - Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

(A) Immunoprecipitation analysis of HEK293T cells transfected with Flag-SQSTM1 or its I431A mutant together with HA-MAVS. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( B ) Luciferase activity driven by IFN - β promoter in HEK293T transfected with HA - MAVS or its 4KR mutant together with Flag - SQSTM1 or its I431A mutant . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-SQSTM1 or its I431A mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( C ) Immunoblot showing of MAVS in HEK293T expressing HA - MAVS or its 4KR mutant together with Flag - SQSTM1 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing HA-MAVS or its 4KR mutant together with Flag-SQSTM1. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( D ) Luciferase activity driven by IFN - β promoter in HEK293T transfected with HA - MAVS or its 4KR mutant together with Flag - NDP52 or its D439R / C443K mutant . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-NDP52 or its D439R/C443K mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( E ) Immunoprecipitation of HEK293T transfected with Flag - MAVS or its 4KR mutant together with Myc - NDP52 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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

(E) Immunoprecipitation analysis of HEK293T cells transfected with Flag-MAVS or its 4KR mutant together with Myc-NDP52. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.