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 .

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

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


E 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 .

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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 .

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

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


A Sec63 - mNeon 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 .

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

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


Time - lapse 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 .

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

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


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

(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 .

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

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


( F ) Rate constants ( kobs ) determined for 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 .

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

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


( A ) 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 .

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


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

(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 .

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

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


C Comparison of meiotic chromosome segregation in 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 .

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

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 .

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

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


G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1 - like - CD 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 .

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

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


A Western blot of whole - 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 .

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

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


D Western blot of FLAG IPs in 3xFLAG - clr4 + cdc2 - as 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O

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


PBMCs from four 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 =

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

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


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O

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


PBMCs from four 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 =

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

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


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O

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


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

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( E ) Dot plot depicting expression of selected ISGs 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 O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( D ) Plot of Log10 average expression of genes showing a Log2 ( fold change ) > 0 . 2 in viral 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( E ) IFN Module Score of viral - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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


( F ) Microscopy 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunoblot showing the levels of the 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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


( A ) Luciferase activity driven by 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


( F ) Immunoprecipitation analysis of the DNP52 - RNF34 interaction in cells infected with VSV . Anti - DNP52 immunoprecipitates were analyzed using immunoblotting with an anti - DNP52 or anti - RNF34 antibody . The WCL was immunoblotted with an anti - MAVS or anti - RNF34 antibody . Cell - based studies were performed independently at least three times with comparable results .

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

(F) Immunoprecipitation analysis of the DNP52-RNF34 interaction in cells infected with VSV. Anti-DNP52 immunoprecipitates were analyzed using immunoblotting with an anti-DNP52 or anti-RNF34 antibody. The WCL was immunoblotted with an anti-MAVS or anti- RNF34 antibody. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoprecipitation analysis of the interaction between Flag - NDP52 and HA - RNF34 in WT or MAVS - / - cells . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA antibody . Cell - based studies were performed independently at least three times with comparable results .

(G) Immunoprecipitation analysis of the interaction between Flag-NDP52 and HA-RNF34 in WT or MAVS-/- cells. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA antibody. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoprecipitation analysis of HEK293T cells transfected with Flag - NDP52 or its D439R / C443K mutant together with HA - MAVS followed by VSV infection . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . Cell - based studies were performed independently at least three times with comparable results .

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

(H) Immunoprecipitation analysis of HEK293T cells transfected with Flag-NDP52 or its D439R/C443K mutant together with HA-MAVS followed by VSV infection. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( I ) Immunoblot showing the levels of the MAVS protein in siCtrl - or siNDP52 - transfected cells expressing Flag - RNF34 or Flag - V . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

(I) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells expressing Flag-RNF34 or Flag-V. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoblot showing the levels of the MAVS protein in siCtrl - or siNDP52 - transfected cells infected with VSV ( MOI = 1 , 12 hrs ) . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

(J) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells infected with VSV (MOI = 1, 12 hrs). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( K ) Immunoprecipitation analysis of the MAVS - LC3 interaction in siCtrl - or siNDP52 - transfected cells infected with VSV . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - LC3 antibody . NDP52 levels were analyzed by performing immunoblotting with an anti - NDP52 antibody . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation analysis of the MAVS-LC3 interaction in siCtrl- or siNDP52-transfected cells infected with VSV. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-LC3 antibody. NDP52 levels were analyzed by performing immunoblotting with an anti-NDP52 antibody. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing MAVS , IRF3 - p , LC3 , TOM20 and HSP60 levels in siCtrl - or siRNF34 - 1 cells infected with VSV and cultured in the presence of Z - VAD ( 50 μM ) for the indicated times . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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

(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing MAVS , LC3 , TOM20 and HSP60 levels in cells transfected with Flag - RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times . Cell - based studies were performed independently at least three times with comparable results .

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

(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


Representative confocal images ( C ) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 cells infected with VSV for the indicated times . Red represents MAVS and green represents NDP52 . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


quantification ( D ) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 cells infected with VSV for the indicated times . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( E ) Transmission electron microscopy images of shCtrl - and shRNF34 - 1 HEK293T cells cultured in the presence or absence of VSV . Arrows indicate mitochondria engulfed in autophagosomes . Double arrows indicate swollen mitochondria with disrupted cristae . M , mitochondria and N , nucleus . 40000 × magnification . Cell - based studies were performed independently at least three times with comparable results .

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(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results.


( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

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(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


Colony formation assay of miRNA mimic transfected - Dgcr8 - / - ESCs . There were 17 miRNAs that decreased the colony forming ability , 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S1 .

Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.


Alkaline phosphatase staining of ESCs after miRNA mimic transfection . There were 21 miRNAs that decreased the AP activity , 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S2 .

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Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.


Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8 - / - ESCs . Error bars indicate s . d . ( n = 3 ) . Full data are shown in Supplementary Fig S3A , B .

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Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.


Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs . 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies , 12 miRNAs mildly affected Oct4 expression , and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies . Representative pictures are shown . Full data are shown in Supplementary Fig S3C .

Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.


AP staining of V6 . 5 ESCs after mimic transfection ( Full data are shown in Supplementary Fig S4B ) .

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AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).


B , C . Northern blot analysis of the expression of miR - 23a , miR - 27a and miR - 24 during EB formation ( B ) and RA induced differentiation of V6 . 5 ESCs ( C ) . The heatmap was representive of the results of quantitative analysis .

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B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.


D , E . The expression of primary miR - 23a ~ 27a ~ 24 - 2 transcript during EB formation ( D ) and RA induced differentiation ( E ) . Error bars indicate s . d . ( n = 3 ) .

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D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).


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F . Q - PCR analysis of miR - 23a , miR - 27a and miR - 24 expression in ESCs ( JM8A3 ) and a variety of C57BL / 6J adult tissues . Data are shown as mean + s . d . ( n = 3 ) .

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F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).


G . Relative protein levels of Oct4 , Sox2 and Nanog in miRNA mimic transfected Dgcr8 - / - ESCs and wild - type ESCs .

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G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.


H , I . Oct4 , Nanog , Foxa2 , Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8 - / - ( H ) and wild - type ESCs ( I ) .

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H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).


Western blot analysis of the expression of target genes in miR - 27a or miR - 24 over - expressed V6 . 5 ESCs . Candidate target genes whose expression was decreased were framed .

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Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.


The relative luciferase activity of the reporter constructs co - transfected with scramble or miR - 27a and miR - 24 mimics . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 . Candidate target genes whose luciferase activity was decreased were framed . PC group was positive reporter control which harbored reverse complementary sequence of miR - 27a or miR - 24 .

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The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.


The relative luciferase activity of the wild - type 3 ′ UTR or mutated 3 ′ UTR reporter co - transfected with scramble or miR - 27a and miR - 24 mimics . Data are shown as mean + s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.


F , G RT - PCR and qRT - PCR analysis of in vivo binding of the miRNAs with their targets . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


H . Western blot analysis of c - Myc expression in siRNA treated V6 . 5 ESCs .

H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.


I . Q - PCR analysis of primary - 23a ~ 27a ~ 24 - 2 , miR - 23a , miR - 27a and miR - 24 expression in c - Myc knocked down ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


K . ChIP - PCR and ChIP - q - PCR analysis of c - Myc binding to the putative sites in the promoter . Error bars indicate s . d . ( n = 3 ) ; * * * indicates P - value < 0 . 001 .

K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.


L . Relative luciferase activity of the wild - type and - 15 binding site mutated promoter in NIH3T3 cells co - transfected with c - Myc or the blank vector ( NC ) . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


M . Relative luciferase activity of wild - type and - 15 binding site mutated promoter in V6 . 5 ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


Validation of the effect of miRNA inhibition in MEFs by q - PCR . Error bars indicate s . d . ( n = 3 ) .

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Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).


AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control , inhibitors of miR - 23a , miR - 27a , miR - 24 and let - 7c respectively .

AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.


Fold increase in Oct4 - GFP - positive cell colonies in the above cells . Data are shown as mean + s . d . ( n = 5 ) ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Morphology of typical Oct4 - GFP - positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors .

Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.


Detection of endogenous and exogenous Oct4 , Sox2 , Klf4 and Nanog expression in iPSCs , ESCs and MEFs by RT - PCR .

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Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.


Immunofluorescence staining of Oct4 , Nanog and SSEA1 of the iPSC colonies .

Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.


Q - PCR analysis of the expression of self - renewal ( Oct4 , Sox2 , Nanog ) and differentiation ( Cdx2 , Foxa2 , Brachyury , Hand1 , Nestin , FGF5 ) relative markers in EBs derived from the iPSCs . Error bars indicate s . d . ( n = 3 ) .

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Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).


Hematoxylin and eosin staining of teratoma derived from the iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) and respiratory epithelium ( Endoderm ) are marked with black arrows .

Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.


Genomic PCR of the specific fragments spanning miR - 23 ~ 27 ~ 24 clusters . WT indicate wild - type ESCs ; DKO indicate miR - 23a ~ 27a ~ 24 - 2 - / - , miR - 23b ~ 27b ~ 24 - 1 - / - ESCs ; KO indicate miR - 23a ~ 27a ~ 24 - 2 - / - ESCs .

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Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.


Real - time PCR analysis of mature miR - 23a / b , miR - 27a / b and miR - 24 expression in wild - type , DKO and KO ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).


AP staining of wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs .

AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.


Real - time PCR analysis of Oct4 , Sox2 and Nanog expression in wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).


Oct4 , Sox2 and Nanog immunofluorescence staining of wild - type and the knockout ESCs .

Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.


Real - time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Beating EB incidence during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Real - time PCR analysis of several cardiac markers expression during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Actinin , ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild - type and knockout ESCs .

Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.