D Representative movie stills of a live - cell imaging experiment illustrating the recruitment of transiently expressed eGFP - ORP1L to lysosomes following 250 µM LLOMe treatment . Cells were pre - treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage ( judged by the decreased number of Lysotracker spots after LLOMe treatment ) . The graph represents quantification of eGFP - ORP1L foci per cell after LLOMe treatment . Error bars denote + / - SEM from n = 3 independent live - cell imaging experiments , > 25 cells were analyzed per experiment .

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

D Representative movie stills of a live-cell imaging experiment illustrating the recruitment of transiently expressed eGFP-ORP1L to lysosomes following 250 µM LLOMe treatment. Cells were pre-treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage (judged by the decreased number of Lysotracker spots after LLOMe treatment). The graph represents quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment.


A Movie stills from a live - cell imaging experiment showing rapid 2xSidM - eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment ( indicated in green ) . Lysosomes were visualized using transiently overexpressed LAMP1 - mCherry ( indicated in red ) . Yellow spots show colocalized PtdIns4P probe ( 2xSidM - eGFP ) and LAMP1 - mCherry after 3 and 10 min of LLOMe treatment . The graph shows the quantification of 2xSidM - eGFP foci per cell after LLOMe treatment . Error bars denote + / - SEM from n = 3 independent live - cell imaging experiments , > 20 cells were analyzed per experiment .

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

A Movie stills from a live-cell imaging experiment showing rapid 2xSidM-eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment (indicated in green). Lysosomes were visualized using transiently overexpressed LAMP1-mCherry (indicated in red). Yellow spots show colocalized PtdIns4P probe (2xSidM-eGFP) and LAMP1-mCherry after 3 and 10 min of LLOMe treatment. The graph shows the quantification of 2xSidM-eGFP foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >20 cells were analyzed per experiment.


B HeLa cells were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h and transiently transfected with the 2xSidM - eGFP probe to monitor PtdIns4P recruitment after LLOMe - treatment by live - cell imaging . Representative movie stills show the dynamics of 2xSidM - eGFP recruitment in control , PI4K2A and PI4K2B depleted cells . The graph represents the quantification of 2xSidM - eGFP foci per cell after LLOMe - treatment . Error bars denote + / - SEM from n = 3 independent live - cell imaging experiments , > 25 cells were analyzed per experiment for each condition .

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

B HeLa cells were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h and transiently transfected with the 2xSidM-eGFP probe to monitor PtdIns4P recruitment after LLOMe-treatment by live-cell imaging. Representative movie stills show the dynamics of 2xSidM-eGFP recruitment in control, PI4K2A and PI4K2B depleted cells. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment for each condition.


C CRISPR - Cas9 mediated knockout of PI4K2A abolishes recruitment of the 2xSidM - eGFP probe ( PtdIns4P ) to damaged lysosomes . Two different clones ( clone 2 and clone 3 ) were tested . The graph represents the quantification of 2xSidM - eGFP foci per cell after LLOMe - treatment . Error bars denote + / - SD from n = 2 independent live - cell imaging experiments , > 20 cells were analyzed per experiment for each condition .

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C CRISPR-Cas9 mediated knockout of PI4K2A abolishes recruitment of the 2xSidM-eGFP probe (PtdIns4P) to damaged lysosomes. Two different clones (clone 2 and clone 3) were tested. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >20 cells were analyzed per experiment for each condition.


D Representative movie stills from live fluorescence microscopy showing reduced eGFP - ORP1L recruitment in PI4K2A knockout cells compared to wild type HeLa cells upon treatment with 250 µM LLOMe . The graph represents the quantification of eGFP - ORP1L foci per cell after LLOMe treatment . Error bars denote + / - SEM from n = 4 independent live - cell imaging experiments , > 80 cells were analyzed per experiment for each condition .

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

D Representative movie stills from live fluorescence microscopy showing reduced eGFP-ORP1L recruitment in PI4K2A knockout cells compared to wild type HeLa cells upon treatment with 250 µM LLOMe. The graph represents the quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >80 cells were analyzed per experiment for each condition.


Representative movie stills from live - cell imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe . Cells were pre - treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe . The graph represents the quantification of bright Lysotracker positive foci per cell . While the decrease in the number of Lysotracker spots is quickly recovering in HeLa cells , there is a severe impairment in lysosomal repair in HeLa - PI4K2A - KO cells .

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

Representative movie stills from live-cell imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe. Cells were pre-treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe. The graph represents the quantification of bright Lysotracker positive foci per cell. While the decrease in the number of Lysotracker spots is quickly recovering in HeLa cells, there is a severe impairment in lysosomal repair in HeLa-PI4K2A-KO cells.


A HeLa cells were co - transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX ( siTSG101 + siALIX ) or with non - targeting siRNA control ( siControl ) , and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM - eGFP . 24 h post - transfection lysosomes were damaged with 250 µM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM - eGFP probe in live - cell imaging experiments . The graph shows quantification of 2xSidM - eGFP foci per cell in cells co - depleted of TSG101 + ALIX and control ( siControl ) cells .

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

A HeLa cells were co-transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX (siTSG101+siALIX) or with non-targeting siRNA control (siControl), and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM-eGFP. 24 h post-transfection lysosomes were damaged with 250 µM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM-eGFP probe in live-cell imaging experiments. The graph shows quantification of 2xSidM-eGFP foci per cell in cells co-depleted of TSG101+ALIX and control (siControl) cells.


A Representative movie stills of a live - cell imaging experiment illustrate faster recruitment of the PtdIns4P probe 2xSidM - eGFP to damaged lysosomes when compared to cholesterol as indicated by the mCherry - D4 probe . The graph shows quantification of 2xSidM - eGFP and mCherry - D4 foci per cell . Error bars denote + / - SD from n = 2 independent live - cell imaging

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

A Representative movie stills of a live-cell imaging experiment illustrate faster recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes when compared to cholesterol as indicated by the mCherry-D4 probe. The graph shows quantification of 2xSidM-eGFP and mCherry-D4 foci per cell. Error bars denote +/- SD from n=2 independent live-cell imaging experiments


D HeLa cells expressing an eGFP - tagged ORP1L mutant incapable of binding to VAP ( mFFAT ) show no accumulation of the cholesterol reporter mCherry - D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype ( wt ) eGFP - ORP1L . The quantification graph shows D4 - mCherry foci per cell . Error bars denote + / - SEM from n = 4 independent live - cell imaging experiments , > 50 cells were analyzed per experiment for each condition .

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D HeLa cells expressing an eGFP-tagged ORP1L mutant incapable of binding to VAP (mFFAT) show no accumulation of the cholesterol reporter mCherry-D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype (wt) eGFP-ORP1L. The quantification graph shows D4-mCherry foci per cell. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >50 cells were analyzed per experiment for each condition.


A Representative movie stills from live - cell imaging experiments indicate recruitment of CHMP4B - eGFP to damaged lysosomes in control and U18666A treated cells . HeLa cells stably expressing CHMP4B - eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe . The graph shows quantification of CHMP4B - eGFP foci per cell . Error bars denote + / - SEM from n = 4 , > 30 cells per experiment for each condition .

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

A Representative movie stills from live-cell imaging experiments indicate recruitment of CHMP4B-eGFP to damaged lysosomes in control and U18666A treated cells. HeLa cells stably expressing CHMP4B-eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of CHMP4B-eGFP foci per cell. Error bars denote +/- SEM from n=4, >30 cells per experiment for each condition.


B Representative immunofluorescence images illustrate Galectin - 3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h . HeLa cells were treated or not ( Control ) with U18666A and + / - LLOMe for the indicated time points , fixed and processed for immunofluorescence microscopy . Endogenous levels of Galectin - 3 were visualized .

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

B Representative immunofluorescence images illustrate Galectin-3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h. HeLa cells were treated or not (Control) with U18666A and +/- LLOMe for the indicated time points, fixed and processed for immunofluorescence microscopy. Endogenous levels of Galectin-3 were visualized.


C Number of Galectin - 3 positive dots per cell was automatically quantified from the dataset described in B . Error bars denote + / - SEM from n = 3 independent experiments . > 225 cells were analyzed per condition , distribution visualized as violin plots .

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C Number of Galectin-3 positive dots per cell was automatically quantified from the dataset described in B. Error bars denote +/- SEM from n=3 independent experiments. >225 cells were analyzed per condition, distribution visualized as violin plots.


D Representative movie stills from live - cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM - eGFP to damaged lysosomes in control but not in the U18666A treated cells . HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe . The graph shows quantification of 2xSidM - eGFP foci per cell .

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

D Representative movie stills from live-cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes in control but not in the U18666A treated cells. HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of 2xSidM-eGFP foci per cell.


A Representative movie stills of a live - cell imaging experiment show recruitment dynamics of transiently expressed eGFP - OSBP in HeLa cells treated with 250 µM LLOMe . The graph shows quantifications of OSBP foci per cell . Error bars denote + / - SEM from n = 5 independent live - cell imaging experiments , > 22 cells per experiment .

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

A Representative movie stills of a live-cell imaging experiment show recruitment dynamics of transiently expressed eGFP-OSBP in HeLa cells treated with 250 µM LLOMe. The graph shows quantifications of OSBP foci per cell. Error bars denote +/- SEM from n=5 independent live-cell imaging experiments, >22 cells per experiment.


D Knock down efficiency of siRNA depletion of OSBP in HeLa cells as detected by Western blot . β - actin used as a loading control .

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D Knock down efficiency of siRNA depletion of OSBP in HeLa cells as detected by Western blot. β-actin used as a loading control.


The violin plot of ACE2 gene expression of all major cell types .

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The violin plot of ACE2 gene expression of all major cell types.


Cell type annotations of all four patients by the tSNE plot . Hepatocyte - cholangiocytes cluster includes potential stem - like cells capable of differentiating into both cell types . B ) tSNE plot colored by

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Cell type annotations of all four patients by the tSNE plot. Hepatocyte-cholangiocytes cluster includes potential stem-like cells capable of differentiating into both cell types. B) tSNE plot colored by patients


Heatmap of marker genes of 16 cell types identified . The red box highlights the cell type specific expression of ACE2 .

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Heatmap of marker genes of 16 cell types identified. The red box highlights the cell type specific expression of ACE2.


The violin plot of ACE2 gene expression across all cell types .

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The violin plot of ACE2 gene expression across all cell types.


Expression of Mpc proteins in mitochondria assessed by SDS - PAGE and immunoblot analysis of crude mitochondrial preparations . The individual untagged Mpc1 ( Mpc1 ) , histidine - tagged Mpc1 ( Mpc1his ) , histidine - tagged Mpc3 ( Mpc3his ) or the Mpc1 / Mpc3 hetero - complex ( Mpc1 / Mpc3his ) were detected with antibodies raised against Mpc1 ( left panel ) or Mpc3 ( right panel ) and are shown with dashed arrows .

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

Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows.


Five micrograms of each affinity - purified Mpc protein were analysed by SDS - PAGE and the bands were visualised by Coomassie Blue stain . Peptide mass finger printing was used to identify the major protein

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Five micrograms of each affinity-purified Mpc protein were analysed by SDS-PAGE and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands


The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM - adduct formation . The thermal denaturation profiles ( left ) were used to calculate the first derivative ( right ) , which provides the apparent melting temperature , indicated with the same colour coding .

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The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding.


The stability of the same samples , as in panel ( D ) , was assessed by NanoDSF . The changes in the 330 nm / 350 nm ratio with temperature ( left ) were used to calculate the first derivative ( right ) .

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The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right).


Nickel - affinity - purified Mpc1 / Mpc3 hetero - complex used for SEC - MALLS analysis , showing a 1 : 1 stoichiometry of the protomers .

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Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.


SEC - MALLS analysis of the hetero - complex . The light scattering trace for Mpc1 / Mpc3 is shown as a black line . The masses of the protein - detergent - lipid complex ( PDL , green ) , the detergent - lipid micelle ( DL , blue ) and the protein ( P , red ) are indicated . Protein fractions across the peak were assessed by SDS - PAGE and visualised by Coomassie Blue staining ( B , inset ) .

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SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).


Nickel - affinity - purified Mpc3 protein used for SEC - MALLS analysis .

Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.


SEC - MALLS analysis of Mpc3 , colour designation as in ( B ) . The protein fractions across the peak ( D , inset ) were assessed as in ( B ) .

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SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).


Time course of pyruvate homo - exchange by the Mpc1 / Mpc3 hetero - complex in liposomes ( n = 8 ) in comparison to empty liposomes ( n = 6 ) at a ΔpH of 1 . 6 .

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Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.


Kinetic analysis of pyruvate homo - exchange at ΔpH of 1 . 6 ( n = 3 ) . The hetero - complex was assayed for initial rates of uptake in the concentration range of 25 - 600 μM . The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats .

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Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.


Time course of pyruvate homo - exchange by the Mpc1 / Mpc3 hetero - complex in physiological pH ( n = 4 ) compared to empty liposomes ( n = 4 ) .

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Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).


In the absence of a ΔpH , the time course of pyruvate homo - exchange was similar for the Mpc1 / Mpc3 proteoliposomes and the empty liposomes ( n = 4 ) .

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In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).


Inhibition of [ 14C ] - pyruvate homo - exchange by UK5099 ( 1 - 100 μM ) , Zaprinast ( 1 - 1000 μM ) , lonidamine ( 10 - 10000 μM ) and 7ACC2 ( 5 - 500 μM ) . Data points represent the mean of three technical replicates of a typical experiment . The IC50 measurements have also been independently replicated , three times for UK5099 Zaprinast and 7ACC2 ( average IC50 9 ± 7 μM , 18 ± 8 μM and 27 ± 13 μM , respectively ) and two times for lonidamine ( average IC50 , 118 ± 24 μM ) .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).


[ 14C ] - pyruvate homo - exchange inhibition by the TZDs , pioglitazone and rosiglitazone ( n = 6 ) .

O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

[14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).


Time course of pyruvate homo - exchange in proteoliposomes at a ΔpH of 1 . 6 was compared for Mpc3 ( n = 4 ) , the Mpc1 / Mpc3 hetero - complex ( n = 6 ) and empty liposomes ( n = 4 ) .

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

Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).


Time course of pyruvate homo - exchange at a ΔpH of 1 . 6 was compared for Mpc1 ( n = 4 ) , Mpc1 / Mpc3 hetero - complex ( n = 4 ) and empty liposomes ( n = 4 ) .

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

Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).


Thermostability analysis via cpm . In detergent solution , Zaprinast ( 250 μM ) increased the thermostability of the Mpc1 / Mpc3 hetero - complex ( black lines ) ( n = 3 ) but not of Mpc3 ( blue lines ) ( n = 3 ) .

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

Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).


Thermostability analysis via nanoDSF . In detergent solution , Zaprinast ( 250 μM ) increased the thermostability of the Mpc1 / Mpc3 hetero - complex ( black lines ) ( n = 3 ) but not of Mpc1 ( orange lines ) ( n = 3 ) .

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

Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).


A ) Image of 8 - 9 day old male control ( w1118 , left ) and chmRNAi ( right ) flies upon ubiquitous chm knockdown ( left ) and the corresponding quantification ( right ) ( N = 6 , Paired t - test was performed for statistical significance ) ; B ) Image of 8 - 9 day old male control ( w1118 , left ) and chmRNAi ( right ) flies upon fat - body specific chm knockdown ( left ) and the corresponding quantification ( right ) ( N = 4 , Paired t - test was performed for statistical significance ) ; C ) Image of 8 - 9 day old male control ( w1118 , left ) and chmRNAi ( right ) flies upon neuron specific chm knockdown ( left ) and the corresponding quantification ( right ) ( N = 3 , Paired t - test was performed for statistical significance ) ; D ) Image of 8 - 9 day old male control ( w1118 , left ) and chmRNAi ( right ) flies upon muscle specific chm knockdown ( left ) and the corresponding quantification ( right ) ( N = 3 , Paired t - test was performed for statistical significance ) Data information : All replicates are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . Paired t - test or Tukey test was performed as indicated and non - significant values are not shown ( * - p < 0 . 05 , * * - p < 0 . 01 , * * * - p < 0 . 001 , * * * * - p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon ubiquitous chm knockdown (left) and the corresponding quantification (right) (N = 6, Paired t-test was performed for statistical significance); B) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon fat-body specific chm knockdown (left) and the corresponding quantification (right) (N = 4, Paired t-test was performed for statistical significance); C) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon neuron specific chm knockdown (left) and the corresponding quantification (right) (N = 3, Paired t-test was performed for statistical significance); D) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon muscle specific chm knockdown (left) and the corresponding quantification (right) (N = 3, Paired t-test was performed for statistical significance) Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). Paired t-test or Tukey test was performed as indicated and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).


A ) Heatmap showing all significant proteins ( FDR < 0 . 05 and Π < 0 . 05 ) between control ( w1118 ) and chmRNAi . Values in the heatmap are scaled raw intensity values . NAs are indicated in grey ;

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

A) Heatmap showing all significant proteins (FDR < 0.05 and Π < 0.05) between control (w1118) and chmRNAi. Values in the heatmap are scaled raw intensity values. NAs are indicated in grey;


A ) Average survival curve of control ( w1118 ) and chmRNAi male flies upon ubiquitous knockdown of chm with da - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ) ; B ) Average survival curve of control ( w1118 ) and chmRNAi male flies upon fatbody knockdown of chm with r4 - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ) ; C ) Average survival curve of control ( w1118 ) and chmRNAi male flies upon neuronal knockdown of chm with elav - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ) ; D ) Average survival curve of control ( w1118 ) and chmRNAi male flies upon muscle knockdown of chm with mef - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ) ; E , F Average survival curve between E ) ethanol ( N = 3 , paired Log - rank test was performed for each biological replicate . p - values ranged between 0 . 2 to 0 . 05 across replicates ) and F ) RU486 ( N = 4 , paired , Log - rank test was performed for each biological replicate with all of them having a p - value < 0 . 05 ) ) and administered control ( w1118 ) and chmRNAi male flies using Act - GS - Gal4 for adult - specific chm knockdown ; G ) Average survival curve of control ( w1118 ) and chmMYST / + male flies ( N = 5 , paired Log - rank test was performed for each biological replicate with all of them having a p - value < 0 . 0001 ) . Data information : All replicates are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . For survival curves , log - rank test was performed for each biological replicate . The displayed p - value is based on all biological replicates .

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

A) Average survival curve of control (w1118) and chmRNAi male flies upon ubiquitous knockdown of chm with da-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); B) Average survival curve of control (w1118) and chmRNAi male flies upon fatbody knockdown of chm with r4-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); C) Average survival curve of control (w1118) and chmRNAi male flies upon neuronal knockdown of chm with elav-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); D) Average survival curve of control (w1118) and chmRNAi male flies upon muscle knockdown of chm with mef-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); E, F Average survival curve between E) ethanol (N = 3, paired Log-rank test was performed for each biological replicate. p-values ranged between 0.2 to 0.05 across replicates) and F) RU486 (N = 4, paired, Log-rank test was performed for each biological replicate with all of them having a p-value < 0.05)) and administered control (w1118) and chmRNAi male flies using Act-GS-Gal4 for adult-specific chm knockdown; G) Average survival curve of control (w1118) and chmMYST/+ male flies (N = 5, paired Log-rank test was performed for each biological replicate with all of them having a p-value < 0.0001). Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed p-value is based on all biological replicates.


D - G Box plot ( left ) and heatmap ( right ) of annotated genes from GO terms of D ) Cellular response to stress ( ANOVA p - value : 6 . 49 x10 - 08 ) ; E ) Response to starvation ( ANOVA p - value : 3 . 09 x10 - 07 ) ; F ) Carbohydrate metabolic process ( ANOVA p - value : 5 . 46 x10 - 07 ) ; G ) Lipid metabolic process ( ANOVA p - value : 7 . 88 x10 - 09 ) ; Box plot shows the average vst normalized read counts of all annotated genes within the GO term averaged over the replicates . Boxes indicate inter - quartile range with central band as the median , central filled dot as the average and the whiskers indicating the maximum and minimum values across 5 biological replicates . Heatmap shows the scaled values of vst normalized read counts in each replicate for all conditions . Data information : All replicates are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . Tukey test was performed and non - significant values are not shown ( * - p < 0 . 05 , * * - p < 0 . 01 , * * * - p < 0 . 001 , * * * * - p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Box plot (left) and heatmap (right) of annotated genes from GO terms of D) Cellular response to stress (ANOVA p-value: 6.49 x10-08); E) Response to starvation (ANOVA p-value: 3.09 x10-07); F) Carbohydrate metabolic process (ANOVA p-value: 5.46 x10-07); G) Lipid metabolic process (ANOVA p-value: 7.88 x10-09); Box plot shows the average vst normalized read counts of all annotated genes within the GO term averaged over the replicates. Boxes indicate inter-quartile range with central band as the median, central filled dot as the average and the whiskers indicating the maximum and minimum values across 5 biological replicates. Heatmap shows the scaled values of vst normalized read counts in each replicate for all conditions. Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). Tukey test was performed and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).


A ) Weight of 8 - 9 day old male chmMYST / + and chmMYST / + ; UAS - chmmyc flies upon weak ( left , N = 5 , paired ) and strong ( right , N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) chm overexpression with arm - Gal4 and da - Gal4 respectively ( Paired t - test was performed ) ; Data information : All are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . Paired t - test was performed for the fly weight data and non - significant values are not shown ( * - p < 0 . 05 , * * - p < 0 . 01 , * * * - p < 0 . 001 , * * * * - p < 0 . 0001 ) .

O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Weight of 8-9 day old male chmMYST/+ and chmMYST/+;UAS-chmmyc flies upon weak (left, N = 5, paired) and strong (right, N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001) chm overexpression with arm-Gal4 and da-Gal4 respectively (Paired t-test was performed); Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). Paired t-test was performed for the fly weight data and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).


B ) Average survival curve of chmMYST / + and chmMYST / + ; UAS - chmmyc male flies upon ubiquitous weak expression of chm with arm - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ; C ) Average survival curve of chmMYST / + and chmMYST / + ; UAS - chmmyc male flies upon ubiquitous strong expression of chm with da - Gal4 ( N = 3 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ; Data information : All are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . For survival curves , log - rank test was performed for each biological replicate . The displayed indicated p - value is based on all biological replicates .

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

B) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon ubiquitous weak expression of chm with arm-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); C) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon ubiquitous strong expression of chm with da-Gal4 (N = 3, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed indicated p-value is based on all biological replicates.


D ) Average survival curve of chmMYST / + and chmMYST / + ; UAS - chmmyc male flies upon fatbody expression of chm with r4 - Gal4 ( N = 4 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ; E ) Average survival curve of control chmMYST / + and chmMYST / + ; UAS - chmmyc male flies upon neuronal expression of chm with elav - Gal4 ( N = 5 , paired , Log - rank test was performed for each biological replicate . All replicates had p - values < 0 . 0001 ) ; F ) Average survival curve of chmMYST / + and chmMYST / + ; UAS - chmmyc male flies upon muscle expression of chm with mef - Gal4 ( N = 5 , paired , Log - rank test was performed for each biological replicate . p - values ranged between 0 . 6 to 0 . 0001 across replicates ) . Data information : All are independent biological replicates and errors bars indicate standard error of the mean ( SEM ) . For survival curves , log - rank test was performed for each biological replicate . The displayed indicated p - value is based on all biological replicates .

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

D) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon fatbody expression of chm with r4-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); E) Average survival curve of control chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon neuronal expression of chm with elav-Gal4 (N = 5, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); F) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon muscle expression of chm with mef-Gal4 (N = 5, paired, Log-rank test was performed for each biological replicate. p-values ranged between 0.6 to 0.0001 across replicates). Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed indicated p-value is based on all biological replicates.


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( D ) GST - tagged RNF34 was subjected to a pull - down 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 .

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

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


( E ) Anti - Flag or IgG immunoprecipitates prepared from 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 .

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

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


( F ) Representative confocal 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 .

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

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


( G ) Immunoblot showing 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 B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD 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 .

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

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


( B ) Immunoprecipitation 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 .

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

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


( C ) Immunoprecipitation 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 B-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD 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 B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-ORGANISM O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD 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 .

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

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

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

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


( A ) Immunoblot showing 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 B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

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

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


( D ) Immunoblot showing 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 B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

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

(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 .

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

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


( D ) Luciferase activity driven by 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 .

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

(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 B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD 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 B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD 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 .

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

(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 B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD 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 .

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

(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 .

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

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

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

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 .

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

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 .

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

(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 .

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

(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.


B | hFwe - Lose mRNA expression is more abundant in elderly people . hFwe - Lose mRNA expression was analyzed by RT - qPCR in 86 lung tissue biopsies taken from non - COVID patients with age between 20 and 82 years . Older patients show a significant upregulation of hFwe - Lose expression . A log - Linear regression model demonstrates a positive correlation between age and hFwe - Lose expression ( R2 = 0 . 13 ; slope confidence interval of 95 % ( CI ) = [ 2 . 0 - 12 . 2 ] ; p - value of the linear regression model < 6 ⋅ 10 - 4 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O B-DISEASE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B | hFwe-Lose mRNA expression is more abundant in elderly people. hFwe-Lose mRNA expression was analyzed by RT-qPCR in 86 lung tissue biopsies taken from non-COVID patients with age between 20 and 82 years. Older patients show a significant upregulation of hFwe-Lose expression. A log-Linear regression model demonstrates a positive correlation between age and hFwe-Lose expression (R2= 0.13; slope confidence interval of 95% (CI) = [2.0-12.2]; p-value of the linear regression model < 6⋅10-4).