Figure 2Nickel - 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 ) . 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 ) .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Nickel-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).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).


Figure 3Time 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 . 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 ) . 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 3Time 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.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).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).


Figure 4Time 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Time 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).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).


Figure 1A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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).


Figure 3A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) 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;


Figure 4A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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.


Figure 5D - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D-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).


Figure 6A ) 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) 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. 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.


Figure 1Results 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 . 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 . ( 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 . ( 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 . ( I ) Results of the ELISA used to determine IFN - β secretion in PBMCs transfected with siCtrl or siRNF34 - 1 oligos and infected with VSV for the indicated times . ELISA data are presented as means ± SEM . Two - tailed Student ' s t - test was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; and * * * , P < 0 . 001 . RT - PCR analysis of the expression of the IL8 , ISG54 and ISG56 ( J ) mRNAs in PBMCs transfected with siCtrl or siRNF34 - 1 oligos and infected with VSV for the indicated times . The results were normalized to the values obtained prior to VSV infection .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Results 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.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.(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.(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.(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.


Figure 2 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.(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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.(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.


Figure 4Immunoprecipitation 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 . ( 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 . ( 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 .

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Figure 4Immunoprecipitation 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.(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.(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.(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.


Figure 6 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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.(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.(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.(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.


Figure 7 ( 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 . 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.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.(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.


Figure 1B | 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 ) . C | hFwe - Lose expression is elevated in lung tissue biopsies from patients with comorbidities . Box plot illustrates an increased expression of hFwe - Lose in lungs of patients with hypertension ( HT ; n = 129 ) , obesity ( n = 45 ) , chronic obstructive pulmonary disease ( COPD n = 51 ) , diabetes ( n = 48 ) , cardio - vascular disease ( CVD ; n = 63 ) versus disease - free control lungs ( n = 42 ) . Patient ` s age is depicted in color . Two - sided Student ' s t - test was performed for each comorbidity ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box . D | hFwe - Lose expression is upregulated in lung tissue of COVID - 19 patients . Box plot illustrates an increased expression of hFwe - Lose in lung tissue of patients diagnosed with COVID - 19 ( n = 11 ) , individuals affected with host comorbidities ( n = 216 ) versus disease - free control lungs ( n = 42 ) . Patient ' s age is depicted in color . Two - sided Student ' s t - test was performed ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box . E | SARS - CoV - 2 - infection manifests histological changes in patients ' lungs as demonstrated by H & E staining . 1 ) For comparison , the normal lung of an elderly individual is shown , containing air - filled empty looking alveolar spaces and thin alveolar septae / gas - exchange membranes . Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested . 2 ) Diffuse ( proliferative ) alveolar damage showing evidence of cell death . An alveolar space filled with desquamated pneumocytes and macrophages , lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II . The still recognizable epithelial lining is detached . Cells and surfactant are lost creating perforations in the alveolar wall , allowing migration of blood cells and fluid to enter inside the alveolar space . The adjacent interstitial space is significantly widened , showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes . On the top and the lower right , dilated and congested capillaries can be seen ( Hematoxylin and Eosin ( H & E ) stain , 200x ) . Abbreviations : a - Alveolar structure destroyed by lymphocytes , desquamated epithelium , as well as extravasated blood ; The alveoli should be empty , but here it is filled with a combination of degenerated cells and fibrin ; b - Next alveoli ; c - Expanded pulmonary interstitium . The interstitium should be as thin as 10um , but here it is 100um ; d - Extravasation of erythrocytes ; e - Lymphocytes ; f - Congested capillaries and arterioles ; g - Multinucleated alveolocyte / pneumocyte type II ; h - Detached epithelium ; i - A histiocyte with a kidney - shaped nucleus at the bottom of the circle has ingested erythrocytes ( left - handed ) , representing the first step of cellular elimination . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 3 ) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining . The adjacent interstitial space shows analogous changes to 2A1 . At the bottom , there is a prominently dilated and congested capillary ( H & E , 200x ) . Abbreviations : a - Alveoli with detached epithelium ; b - Next alveoli ; c - E Extravasation of erythrocytes ; d - Expanded interstitium ; e - Lymphocytes ; f - Multinucleated pneumocyte type II . 4 ) Diffuse alveolar damage showing massive extravasation of fibrin ( homogeneous eosinophilic material in the center of the alveolar space ) . The lining pneumocytes are almost all apoptotic / necrotic . The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris ( H & E , 200x ) . Abbreviations : a - Alveola ; b - Congested capillaries ; c - Fibrin ; d - Remnants of degenerated / dying epithelium ; e - Expanded interstitium ; f - Cellular debris consisting of macrophages , detached epithelial cells and lymphocytes ; g - Fully degenerated / lacking epithelial coverage within the alveoli . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 5 ) Immunohistochemical stain ( IHC ) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes / lung interstitium leading to obstruction of the microcirculation ( IHC for fibrin , 200x ) . Abbreviations : a - All small alveolar septal capillaries are filled out with worm - like fibrin thrombi hampering / obstructing circulation ; b - Normal interstitium . F | Healthy lungs show very low cleaved caspase - 3 positive cells . IHC staining for cleaved caspase - 3 ( 20x ) . G | Expression of cleaved ( active ) caspase - 3 yielding apoptotic and pre - apoptotic cells : IHC for cleaved ( active ) caspase - 3 showing a brown nuclear staining signal in respective cells . 31 . 5 % cells were found to be caspase positive , 11 . 7 % with high caspase - 3 positivity and another 19 . 8 % with low . These cells are mainly located in the interstitium , but also in some alveolar epithelial and endothelial compartments . In the lower right , the edge of an alveolar space containing several ( pre - ) apoptotic pneumocytes is seen , while in the upper left a completely denuded / deepithelialized alveolus with two apoptotic remnants is observable ( IHC for cleaved caspase - 3 , 20x ) . Abbreviations : a - Apoptotic large mononuclear cells ; b - Apoptotic alveolocyte / pneumocyte ; i - Amplification of apoptotic alveolocyte / pneumocyte . As shown in the round inlets of 1E2 and 1E4 , homogeneous dark nuclear condensation up to a size of 2μm ( karyopyknosis ) , homogeneous pinkish - greyish nuclear condensation , large nuclear inclusion with marginalization of the condensed chromatin , coarse nuclear angulation , nose - like / polar body - like nuclear protrusions , the latter two being karyorrhectic debris , were all morphologically considered evidence of apoptosis . This has been correlated with and was reflected by the results of the cleaved caspase - 3 staining on step sections . All cells were counted on the 20X fields for n = 42 disease - free and n = 3 COVID - 19 autopsy patients . The expression of hFwe - Lose in COVID - 19 patients is significantly higher than in disease - free individuals , regardless of cleaved caspase - 3 staining ( Cas POS : p < 0 . 004 ; Cas NEG : p < 0 . 006 ) . In COVID - 19 patients we observed a significantly higher expression of hFwe - Lose in sections with positive cleaved caspase - 3 staining ( p < 0 . 004 ) . Two - sided Student ' s t - test was performed ( compared to disease - free samples and NEG samples , respectively ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B | 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).C | hFwe-Lose expression is elevated in lung tissue biopsies from patients with comorbidities. Box plot illustrates an increased expression of hFwe-Lose in lungs of patients with hypertension (HT; n = 129), obesity (n = 45), chronic obstructive pulmonary disease (COPD n = 51), diabetes (n = 48), cardio-vascular disease (CVD; n = 63) versus disease-free control lungs (n = 42). Patient`s age is depicted in color. Two-sided Student's t-test was performed for each comorbidity (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.D | hFwe-Lose expression is upregulated in lung tissue of COVID-19 patients. Box plot illustrates an increased expression of hFwe-Lose in lung tissue of patients diagnosed with COVID-19 (n = 11), individuals affected with host comorbidities (n = 216) versus disease-free control lungs (n = 42). Patient's age is depicted in color. Two-sided Student's t-test was performed (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.E | SARS-CoV-2-infection manifests histological changes in patients' lungs as demonstrated by H&E staining. 1)For comparison, the normal lung of an elderly individual is shown, containing air-filled empty looking alveolar spaces and thin alveolar septae/gas-exchange membranes. Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested. 2) Diffuse (proliferative) alveolar damage showing evidence of cell death. An alveolar space filled with desquamated pneumocytes and macrophages, lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II. The still recognizable epithelial lining is detached. Cells and surfactant are lost creating perforations in the alveolar wall, allowing migration of blood cells and fluid to enter inside the alveolar space. The adjacent interstitial space is significantly widened, showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes. On the top and the lower right, dilated and congested capillaries can be seen (Hematoxylin and Eosin (H&E) stain, 200x). Abbreviations: a- Alveolar structure destroyed by lymphocytes, desquamated epithelium, as well as extravasated blood; The alveoli should be empty, but here it is filled with a combination of degenerated cells and fibrin; b- Next alveoli; c- Expanded pulmonary interstitium. The interstitium should be as thin as 10um, but here it is 100um; d- Extravasation of erythrocytes; e- Lymphocytes; f- Congested capillaries and arterioles; g- Multinucleated alveolocyte/pneumocyte type II; h- Detached epithelium; i- A histiocyte with a kidney-shaped nucleus at the bottom of the circle has ingested erythrocytes (left-handed), representing the first step of cellular elimination. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 3) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining. The adjacent interstitial space shows analogous changes to 2A1. At the bottom, there is a prominently dilated and congested capillary (H&E, 200x). Abbreviations: a- Alveoli with detached epithelium; b- Next alveoli; c- E Extravasation of erythrocytes; d- Expanded interstitium; e- Lymphocytes; f- Multinucleated pneumocyte type II. 4) Diffuse alveolar damage showing massive extravasation of fibrin (homogeneous eosinophilic material in the center of the alveolar space). The lining pneumocytes are almost all apoptotic/necrotic. The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris (H&E, 200x). Abbreviations: a- Alveola; b- Congested capillaries; c- Fibrin; d- Remnants of degenerated / dying epithelium; e- Expanded interstitium; f- Cellular debris consisting of macrophages, detached epithelial cells and lymphocytes; g- Fully degenerated / lacking epithelial coverage within the alveoli. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 5) Immunohistochemical stain (IHC) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes/lung interstitium leading to obstruction of the microcirculation (IHC for fibrin, 200x). Abbreviations: a- All small alveolar septal capillaries are filled out with worm-like fibrin thrombi hampering / obstructing circulation; b- Normal interstitium.F | Healthy lungs show very low cleaved caspase-3 positive cells. IHC staining for cleaved caspase-3 (20x). G | Expression of cleaved (active) caspase-3 yielding apoptotic and pre-apoptotic cells: IHC for cleaved (active) caspase-3 showing a brown nuclear staining signal in respective cells. 31.5% cells were found to be caspase positive, 11.7% with high caspase-3 positivity and another 19.8% with low. These cells are mainly located in the interstitium, but also in some alveolar epithelial and endothelial compartments. In the lower right, the edge of an alveolar space containing several (pre-)apoptotic pneumocytes is seen, while in the upper left a completely denuded/deepithelialized alveolus with two apoptotic remnants is observable (IHC for cleaved caspase-3, 20x). Abbreviations: a- Apoptotic large mononuclear cells; b-Apoptotic alveolocyte / pneumocyte; i- Amplification of apoptotic alveolocyte / pneumocyte. As shown in the round inlets of 1E2 and 1E4, homogeneous dark nuclear condensation up to a size of 2μm (karyopyknosis), homogeneous pinkish-greyish nuclear condensation, large nuclear inclusion with marginalization of the condensed chromatin, coarse nuclear angulation, nose-like/polar body-like nuclear protrusions, the latter two being karyorrhectic debris, were all morphologically considered evidence of apoptosis. This has been correlated with and was reflected by the results of the cleaved caspase-3 staining on step sections. All cells were counted on the 20X fields for n = 42 disease-free and n = 3 COVID-19 autopsy patients. The expression of hFwe-Lose in COVID-19 patients is significantly higher than in disease-free individuals, regardless of cleaved caspase-3 staining (Cas POS: p < 0.004; Cas NEG: p < 0.006). In COVID-19 patients we observed a significantly higher expression of hFwe-Lose in sections with positive cleaved caspase-3 staining (p < 0.004). Two-sided Student's t-test was performed (compared to disease-free samples and NEG samples, respectively), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.