Cell survival analysis of the indicated HeLa cells . 7 × 105 cells in one well of 6 - well plate were treated with ActD ( 200 nM ) for 4 hours or Doxo ( 50 nM ) for 2 hours and subsequently cultured in normal medium for 21 days . Surviving colonies were stained by 0 . 1 % ( W / V ) methylene blue . Representative images and quantifications of the surviving colony numbers are shown . Data information : Data are shown as mean ± SE from three biological repeats G ) . Statistics : one - way ANOVA ( G ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of BH3 - domain containing proteins in the indicated HeLa cells treated with Doxo ( 10 μM ) . Three proteins with enhanced level in ∆ UBXD8 cells ( Noxa , Bik , and Bnip3 ) are highlighted in red .

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Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.


Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 blot and quantitative analysis of Noxa degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats C , Statistics : two - tailed unpaired Student ' s t - test C , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

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Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats C, Statistics: two-tailed unpaired Student's t-test C, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot ( D , analysis of the indicated HeLa cells .

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Western blot (D, analysis of the indicated HeLa cells.


Western blot F ) analysis of the indicated HeLa cells .

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Western blot F) analysis of the indicated HeLa cells.


Representative images of the mitophagy events in the indicated HeLa cells . mtKeima emission excited by 405 nm laser is shown as green ; mtKeima emission excited by 561 nm laser is shown as red . Mitolysosomes are pointed by white arrows . Scale bar , 5 μm .

O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O 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-SUBCELLULAR O O O O O O O O O O O O

Representative images of the mitophagy events in the indicated HeLa cells. mtKeima emission excited by 405 nm laser is shown as green; mtKeima emission excited by 561 nm laser is shown as red. Mitolysosomes are pointed by white arrows. Scale bar, 5 μm.


C - F . Quantitative analysis of mitophagy events in the indicated HeLa cells . Only cells with mitophagy were selected to quantify mitolysosome numbers ( D , F ) . Data information : Data are shown as mean ± SE from three biological repeats ( C - F , Statistics : two - tailed unpaired Student ' s t - test ( C - F , ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Quantitative analysis of mitophagy events in the indicated HeLa cells. Only cells with mitophagy were selected to quantify mitolysosome numbers (D, F). Data information: Data are shown as mean ± SE from three biological repeats (C-F, Statistics: two-tailed unpaired Student's t-test (C-F, ; **P < 0.01, ***P < 0.001.


I - K , Representative images ( I ) and quantitative analysis of mitophagy events ( J , K ) in the indicated HeLa cells . White arrows point to mitolysosomes . Scale bar , 5 μm . Data information : Data are shown as mean ± SE from three biological repeats J , K , Statistics : two - tailed unpaired Student ' s t - test J , K ) ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K, Representative images (I) and quantitative analysis of mitophagy events (J, K) in the indicated HeLa cells. White arrows point to mitolysosomes. Scale bar, 5 μm. Data information: Data are shown as mean ± SE from three biological repeats J, K, Statistics: two-tailed unpaired Student's t-test J, K); **P < 0.01, ***P < 0.001.


A . Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation . The gray scale indicates the density of dots in a given area of the chart . The dotted contours represent the 0 . 5 , 0 . 75 and 0 . 95 probability contours of the 240 WT replicates . The Pearson correlation is ρ = 0 . 65 , with a 95 % CI = [ 0 . 63 , 0 . 66 ] . The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time . Red dots highlight

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strains


C . Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain . The cumulative distributions of the fraction of cells with two nucleoids ( blue ) and of the fraction of cells with a constriction degree above 0 . 15 ( red ) were plotted against cell age . Cell age was calculated according to the rank of each cell based on their cell length with the formula $ \ text { age } _ { i } \ left ( F \ right ) = \ - \ frac { ln ( 1 - \ frac { F } { 2 } ) } { ln ( 2 ) } $ , where F represents the fraction of cells with a cell length equal or below the length of cell i ( Wold et al , 1994 ) . D . Same plots as in C for three strains clustering in island 8 with the ΔmatP strain . The WT curves shown in C were plotted in gray for comparison . E . Same plots as in C for two island - 8 strains , ΔycjV and ΔhslU , and the ΔhslV strain , which does not partition in island 8 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain. The cumulative distributions of the fraction of cells with two nucleoids (blue) and of the fraction of cells with a constriction degree above 0.15 (red) were plotted against cell age. Cell age was calculated according to the rank of each cell based on their cell length with the formula $\text{age}_{i}\left( F \right) = \ - \frac{ln(1 - \frac{F}{2})}{ln(2)}$, where F represents the fraction of cells with a cell length equal or below the length of cell i (Wold et al, 1994). D. Same plots as in C for three strains clustering in island 8 with the ΔmatP strain. The WT curves shown in C were plotted in gray for comparison. E. Same plots as in C for two island-8 strains, ΔycjV and ΔhslU, and the ΔhslV strain, which does not partition in island 8.


B . The cumulative distribution of the proportion of constricting cells for the ftsA * mutant and its parent were plotted against cell age . We measured the degree of constriction of all cells using Oufti . For each strain , we had two independently acquired image sets ( n = 496 and 1 , 168 cells for the WT replicates and n = 692 and 1 , 506 cells for the ftsA * replicates ) . The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p - value of 0 . 01 using a Kruskal - Wallis multi - comparison test . Moreover , Bonferroni - corrected post - hoc pairwise tests did not allow a distinction between WT and ftsA * samples .

O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O 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-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O

B. The cumulative distribution of the proportion of constricting cells for the ftsA* mutant and its parent were plotted against cell age. We measured the degree of constriction of all cells using Oufti. For each strain, we had two independently acquired image sets (n = 496 and 1,168 cells for the WT replicates and n = 692 and 1,506 cells for the ftsA* replicates). The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p-value of 0.01 using a Kruskal-Wallis multi-comparison test. Moreover, Bonferroni-corrected post-hoc pairwise tests did not allow a distinction between WT and ftsA* samples.


C . Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only . The contour lines represent the 0 . 5 , 075 and 0 . 95 probability envelopes of the 240 independent WT replicates . The gray color levels indicate the density of points in the vicinity of each strain . The ∆ mraZ strain discussed in the text is highlighted in red .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O

C. Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only. The contour lines represent the 0.5, 075 and 0.95 probability envelopes of the 240 independent WT replicates. The gray color levels indicate the density of points in the vicinity of each strain. The ∆mraZ strain discussed in the text is highlighted in red.


( A ) Western blotting of AXL and OPCML protein levels in AXL - expressing , OPCML - null SKOV3 and PEA1 cell lines transduced with OPCML " O " or control Empty vector " E "

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O

(A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E"


( B ) Mammalian 2 - Hybrid assay between OPCML and AXL 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

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mammalian 2-Hybrid assay between OPCML and AXL 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


( C ) Western blotting of the OPCML - GST pull down . Input : 1 / 20 of SKOV3 - Empty whole cell

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O

(C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate


( D ) Western blotting of the anti - AXL immunoprecipitation . Input : 1 / 100 of SKOV3 - OPCML whole cell lysate . IP : Immunoprecipitated protein , IB : Immunoblotted

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY

(D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( E ) Immunostaining of OPCML ( red ) and AXL ( green ) in SKOV3 - OPCML and PEA1 - OPCML cells . Scale bar = 10μm Data i M , ( E ) was performed once due to the limited numbers of primary cells ;

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(E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;


( 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

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and PEA1 cells transduced with empty vector " E " or OPCML " O " . Scale bar =

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O B-GENEPROD 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


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

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD 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


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

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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 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

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 -

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O O O O B-CELL_LINE 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 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

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(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 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 )

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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 ) 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

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(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 ) and quantified , n =

O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY 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 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

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cells stimulated with Gas6 as indicated . Input : 1 / 50 of whole cell lysate from SKOV3 - OPCML cells . IP : Immunoprecipitated protein , IB : Immunoblotted

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(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 cells stimulated with

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O B-GENEPROD O O O

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


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

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


( 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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD

(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 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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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

O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 " 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

O O O B-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ±

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O 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 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 )

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O 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 cells treated with MBCD and separated into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD

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

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD 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 and SKOV3 - OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot . GAPDH was used as loading

O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD 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 and pERK in SKOV3 - EMPTY " E " and SKOV3 - OCPML " O " , treated with MBCD and no treatment control , normalised to GAPDH , from ( D )

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

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE O B-GENEPROD 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 " 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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD 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 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 =

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O 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 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 .

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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 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 .

O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 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 .

O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 and Rtn1 - mCherry images of ∆ lnp1 and ∆ sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h .

O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O B-SUBCELLULAR O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O 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 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 O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O 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 ) 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 .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O 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 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 .

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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 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 .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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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 , 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 .

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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 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 .

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(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 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 .

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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 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 .

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(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 the WT and the E239K mutant . The arrowheads represent the end positions of the WT and E239K .

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(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 ) 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 .

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(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 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 .

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(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 ) 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 .

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(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 .

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


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

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


( 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 .

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(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 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 .

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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 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 .

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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 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 .

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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 - 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 .

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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 levels of Clr4 in the indicated homothallic strains .

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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 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 .

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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 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 .

O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 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 =

B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE 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 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 .

O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 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 =

B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE 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 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 .

O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE 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.


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 ) .

O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O 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 and ISG15 mRNA expression in the indicated conditions . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O 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 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 .

O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD 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 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 .

O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 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 .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY 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 - 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 .

O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM 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-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O

(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 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 .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-ORGANISM 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM 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 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 .

O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.