figf7 ( a - d ) Analysis of HeLa cells stably expressing green fluorescent protein - LC3 , infected with S . Typhimurium . ( a ) Confocal micrographs of cells stained 1 h after infection with NDP52 antiserum . Scale bar , 10 μm ; arrowheads , bacteria shown in insets . ( b ) Colocalization of NDP52 with LC3 - positive bacteria . ( c , d ) Counts of LC3 - positive S . Typhimurium 1 h after infection in cells transfected with the indicated siRNAs . In c , cells were stained for ubiquitin ( Ub ) ; in d , red fluorescence protein ( RFP ) - expressing S . Typhimurium were used . Means of duplicate counts from four independent experiments ; * P 0 . 05 , Student ' s t - test . ( e ) Binding of NDP52 to GST - LC3 and GST - ubiquitin . Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E . coli expressing NDP52 . Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS - PAGE . ( f ) Normalized ratio of luciferase - LC3 binding to beads . The indicated purified GST proteins coupled to beads were incubated with bacterially expressed LC3 fused to luciferase and the indicated volumes of lysed E . coli expressing NDP52 . Data in a , b , e and f are representative of at least two independent experiments .

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figf7(a-d) Analysis of HeLa cells stably expressing green fluorescent protein-LC3, infected with S. Typhimurium. (a) Confocal micrographs of cells stained 1 h after infection with NDP52 antiserum. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b) Colocalization of NDP52 with LC3-positive bacteria. (c,d) Counts of LC3-positive S. Typhimurium 1 h after infection in cells transfected with the indicated siRNAs. In c, cells were stained for ubiquitin (Ub); in d, red fluorescence protein (RFP)-expressing S. Typhimurium were used. Means of duplicate counts from four independent experiments; *P 0.05, Student's t-test.(e) Binding of NDP52 to GST-LC3 and GST-ubiquitin. Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E. coli expressing NDP52. Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS-PAGE.(f) Normalized ratio of luciferase-LC3 binding to beads. The indicated purified GST proteins coupled to beads were incubated with bacterially expressed LC3 fused to luciferase and the indicated volumes of lysed E. coli expressing NDP52. Data in a,b,e and f are representative of at least two independent experiments.


Figure 1 ( C ) High - performance thin - layer chromatography ( HPTLC ) profile of HeLa cells pulsed for 2 hours with [ 3H ] - sphingosine and chased for 24 hours . The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL .

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Figure 1(C) High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 hours with [3H]-sphingosine and chased for 24 hours. The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL.


Figure 2 ( A ) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [ 3H ] - sphingosine pulse - chase assay . GSL levels are expressed as fold changes with respect to control . CERT and FAPP2 knockdowns ( blue bars ) were used as controls . ( D ) Control and GRASP55KO cells were processed for Cy3 - conjugated Shiga Toxin ( ShTxB ) and Alexa488 - conjugated Cholera Toxin ( ChTxB ) staining followed by flow cytometry analysis . Mean fluorescence intensity was measured and represented .

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Figure 2(A) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls.(D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented.


Figure 3 ( C ) Control and GRASP55 KO cells were transfected for 16 hours with GCS - HA or LCS - HA and processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) in case of GCS - HA and anti - HA antibody ( 15 - nm gold particles ) and anti - GM130 antibody ( 10 - nm gold particles ) in case of LCS - HA . Representative images of the distribution of GCS - HA and LCS - HA are shown . Scale bar 200nm . Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling .

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Figure 3(C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling.


Figure 4 ( A ) HeLa cells co - transfected with indicated HA tagged enzymes ( GCS , LCS , GM3S and Gb3S ) and GRASP55 - GFP were lysed , immunoprecipitated with anti - GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting ( IB ) with anti - HA antibody . I represents 5 % on the input lysate , U 5 % of the unbound fraction and IP the immunoprecipitate . Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA - tagged enzymes ( E ) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS ( WT and Δ3C ) and indicated purified GST - tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti - GST tag antibody .

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Figure 4(A) HeLa cells co-transfected with indicated HA tagged enzymes (GCS, LCS, GM3S and Gb3S) and GRASP55-GFP were lysed, immunoprecipitated with anti-GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting (IB) with anti-HA antibody. I represents 5% on the input lysate, U 5% of the unbound fraction and IP the immunoprecipitate. Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA-tagged enzymes(E) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS (WT and Δ3C) and indicated purified GST-tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti-GST tag antibody.


Figure 5 ( A - C ) Control and GRASP55 KO ( # 2 ) cells were transfected for 16 hours with the indicated HA - tagged enzymes and processed for cryo - immunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) . Representative images of the distribution of HA - tagged enzymes are shown . Red circles indicate GM130 labelling . Arrow heads represent the peri - Golgi vesicles . Scale bar 150 nm . Quantification of the distribution of enzymes in vesicles represented as normalized linear density ( F ) HeLa cells co - transfected with HA - tagged GCS and GRASP55 - GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GFP antibody ( 15 - nm gold particles ) . Representative images of the distribution of GCS and GRASP55 - GFP are shown . Red arrowhead indicates the clathrin - coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS - HA in TGN . Scale bar 150 nm . Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles .

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Figure 5(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density(F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles.


Figure 6 ( A Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . Total GSL levels were quantified and expressed as fold changes with respect to control ( A ) . B ) Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . The GM / Gb ratio was calculated and represented ( B ) . ( F ) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO ( # 2 ) clone before subjecting to [ 3H ] - sphingosine pulse chase assay . GSLs were quantified and represented as fold change with respect to control .

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Figure 6(A Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A).B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). The GM/Gb ratio was calculated and represented (B).(F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control.


Figure 1B HCT116 and SW480 cells with GDH1 knockdown were incubated under normoxia and hypoxia , respectively . Then , cell viability was assessed by trypan blue staining . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates ( B Statistics : two - way ANOVA with Tukey ' s HSD post hoc test ( B ) D The cells in Figure 1B were collected after incubation under normoxia or hypoxia induction for 6 hours , and the HIF1α protein level was measured via immunoblot analysis . F HIF1A gene transcription was slightly impaired after GDH1 depletion . The cells in Figure 1B were collected after incubation under normoxia or hypoxia , and the HIF1A mRNA level was analysed via quantitative real - time PCR ( qRT ‒ PCR ) . ( n = 3 ; ns , not significant ) . Data information : Data are mean ± SD from the biological replicates Statistics : unpaired two - tailed student ' s t - testH Tumour formation study . HCT116 - shNT and HCT116 - shGDH1 cells were injected into the left groin of nude mice . Solid tumours were stained with an antibody against Ki67 ( Scale bars : 50 μm ) . ( n = 3 ) ( H ) . Data information : Data are mean ± SD from the biological replicates Statistics : unpaired two - tailed student ' s t -

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Figure 1B HCT116 and SW480 cells with GDH1 knockdown were incubated under normoxia and hypoxia, respectively. Then, cell viability was assessed by trypan blue staining. (n=3). Data information: Data are mean ± SD from the biological replicates (B Statistics: two-way ANOVA with Tukey's HSD post hoc test (B)D The cells in Figure 1B were collected after incubation under normoxia or hypoxia induction for 6 hours, and the HIF1α protein level was measured via immunoblot analysis.F HIF1A gene transcription was slightly impaired after GDH1 depletion. The cells in Figure 1B were collected after incubation under normoxia or hypoxia, and the HIF1A mRNA level was analysed via quantitative real-time PCR (qRT‒PCR). (n=3; ns, not significant). Data information: Data are mean ± SD from the biological replicates Statistics: unpaired two-tailed student's t-testH Tumour formation study. HCT116-shNT and HCT116-shGDH1 cells were injected into the left groin of nude mice. Solid tumours were stained with an antibody against Ki67 (Scale bars: 50 μm). (n=3) (H). Data information: Data are mean ± SD from the biological replicates Statistics: unpaired two-tailed student's t-test


Figure 2 ( E ) H & E staining of colon cancer tumours from GDH1IEC + / + and GDH1IEC - / - mice ( scale bar , 200 μm ) . F F4 / 80 staining in a noninflamed portion of the colon after AMO / DSS treatment ( scale bar , 50 μm ) . G ZO - 1 staining in colon sections of mice treated with sequential AMO / DSS treatment ( scale bar , 200 μm ) . I Survival percentages of GDH1IEC + / + mice ( n = 11 ) and GDH1IEC - / - mice ( n = 9 ) during progression of the inflammatory cancer switch . Data information : Statistics : log - rank test ( I ) . K Both HIF1α protein levels and AMPK activity were assessed in the intestine of GDH1IEC - / - mice after AOM / DSS treatment using the indicated antibodies .

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Figure 2(E) H&E staining of colon cancer tumours from GDH1IEC+/+ and GDH1IEC-/- mice (scale bar, 200 μm). F F4/80 staining in a noninflamed portion of the colon after AMO/DSS treatment (scale bar, 50 μm). G ZO-1 staining in colon sections of mice treated with sequential AMO/DSS treatment (scale bar, 200 μm).I Survival percentages of GDH1IEC+/+ mice (n=11) and GDH1IEC-/- mice (n=9) during progression of the inflammatory cancer switch. Data information: Statistics: log-rank test (I).K Both HIF1α protein levels and AMPK activity were assessed in the intestine of GDH1IEC-/- mice after AOM/DSS treatment using the indicated antibodies.


Figure 3C GDH1 - FLAG in ( B ) was examined using antibodies against pan - lysine acetylation and methylation and pan - serine / threonine and tyrosine phosphorylation . E HCT116 or SW480 cells containing WT or GDH1 with K503 / K527R mutations were incubated under normoxia and hypoxia , respectively . GDH1 - FLAG was enriched , and an antibody against pan - lysine acetylation was used to test GDH1 - lysine acetylation . H GDH1 - depleted cell lines were constructed and then rescued with the expression of wild - type GDH1 or K503 / K527R mutant GDH1 .

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Figure 3C GDH1-FLAG in (B) was examined using antibodies against pan-lysine acetylation and methylation and pan-serine/threonine and tyrosine phosphorylation.E HCT116 or SW480 cells containing WT or GDH1 with K503/K527R mutations were incubated under normoxia and hypoxia, respectively. GDH1-FLAG was enriched, and an antibody against pan-lysine acetylation was used to test GDH1-lysine acetylation.H GDH1-depleted cell lines were constructed and then rescued with the expression of wild-type GDH1 or K503/K527R mutant GDH1.


Figure 4B Immunoblot analysis of GDH1 - FLAG levels at different time points . D The K503Q mutation reduces GDH1 activity leading to αKG production while enhancing the enzymatic activity leading to Glu production . His - tagged GDH1 was purified from E . coli . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates ( C - G Statistics : one - way ANOVA with Tukey ' s HSD post hoc testF HCT116 or SW480 cells were depleted of endogenous GDH1 and rescued with SFB - tagged rGDH1 WT , K503Q or K503R . rGDH1 - SFB proteins were enriched using M2 - FLAG beads to determine the Vmax of αKG production ( F ) . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates Statistics : one - way ANOVA with Tukey ' s HSD post hoc testITC assays were performed with precipitated His - GDH1 proteins and αKG ( I ) . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates I ) . Statistics : one - way ANOVA with Tukey ' s HSD post hoc test I ) .

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Figure 4B Immunoblot analysis of GDH1-FLAG levels at different time points.D The K503Q mutation reduces GDH1 activity leading to αKG production while enhancing the enzymatic activity leading to Glu production. His-tagged GDH1 was purified from E. coli. (n=3). Data information: Data are mean ± SD from the biological replicates (C-G Statistics: one-way ANOVA with Tukey's HSD post hoc testF HCT116 or SW480 cells were depleted of endogenous GDH1 and rescued with SFB-tagged rGDH1 WT, K503Q or K503R. rGDH1-SFB proteins were enriched using M2-FLAG beads to determine the Vmax of αKG production (F). (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testITC assays were performed with precipitated His-GDH1 proteins and αKG (I). (n=3). Data information: Data are mean ± SD from the biological replicates I). Statistics: one-way ANOVA with Tukey's HSD post hoc test I).


Figure 5A , B Immunoblot of the HIF1α protein level under hypoxia for 6 hours when enzymes related to the αKG level were overexpressed in cells , including GDH1 ( A ) . Relative αKG levels in contrast to the control ( B ) . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates ( B Statistics : one - way ANOVA with Tukey ' s HSD post hoc test ( BE Immunofluorescence analysis of GDH1 - FLAG and HA - EGLN1 was performed to assess their colocalization under normoxia and hypoxia . I EGLN1 activity test . His - GDH1 with K503 or K527 acetylation ( AcK503 or AcK527 ) and mimic acetylation at K503 or K527 ( K503Q or K527Q ) were purified from E . coli . Then , His - GDH1 was incubated for 1 hour with HA - EGLN1 ( I ) . EGLN1 activity was tested by converting αKG to succinate . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates Statistics : one - way ANOVA with Tukey ' s HSD post hoc testDetection of HIF1α protein stability ( L ) in the indicated cell lines under hypoxia . ( n = 3 ) . the mRNA expression levels of the HIF1α target genes GLUT1 , HK1 , PGK1 and CCND1 ( M ) in the indicated cell lines under hypoxia . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates Statistics : one - way ANOVA with Tukey ' s HSD post hoc testO Detection of HIF1α protein stability in the indicated cell lines under normoxia . P HIF1α transactivation was examined in the indicated cell lines under hypoxia with or without treatment with the HIF1α transactivation inhibitor 2 - MeOE2 ( 1 μM ) for 12 hours . ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates P ) . Statistics : one - way ANOVA with Tukey ' s HSD post hoc test , P )

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Figure 5A, B Immunoblot of the HIF1α protein level under hypoxia for 6 hours when enzymes related to the αKG level were overexpressed in cells, including GDH1 (A). Relative αKG levels in contrast to the control (B). (n=3). Data information: Data are mean ± SD from the biological replicates (B Statistics: one-way ANOVA with Tukey's HSD post hoc test (BE Immunofluorescence analysis of GDH1-FLAG and HA-EGLN1 was performed to assess their colocalization under normoxia and hypoxia.I EGLN1 activity test. His-GDH1 with K503 or K527 acetylation (AcK503 or AcK527) and mimic acetylation at K503 or K527 (K503Q or K527Q) were purified from E. coli. Then, His-GDH1 was incubated for 1 hour with HA-EGLN1(I). EGLN1 activity was tested by converting αKG to succinate. (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testDetection of HIF1α protein stability (L) in the indicated cell lines under hypoxia. (n=3).the mRNA expression levels of the HIF1α target genes GLUT1, HK1, PGK1 and CCND1 (M) in the indicated cell lines under hypoxia. (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testO Detection of HIF1α protein stability in the indicated cell lines under normoxia.P HIF1α transactivation was examined in the indicated cell lines under hypoxia with or without treatment with the HIF1α transactivation inhibitor 2-MeOE2 (1 μM) for 12 hours. (n=3). Data information: Data are mean ± SD from the biological replicates P). Statistics: one-way ANOVA with Tukey's HSD post hoc test , P)


Figure 6C GDH1 - K503 or GDH1 - K527 acetylation occurred in both the cytosol and nucleus . D , The levels of both AcK503 and AcK527 were analysed when cells were transfected with small interfering RNA ( siRNA ) targeting TIP60 , PCAF , CBP or p300 ( D ) F HCT116 and SW480 cells were transfected with or without FLAG - p300 . The p300 - associated proteins were enriched with M2 - FLAG beads and subjected to immunoblot analysis using antibodies against GDH1 , GDH1 - K503 or GDH1 - K527 acetylation . K The HIF1A gene was transiently knocked down , and HCT116 cells were cotransfected with FLAG - p300 and HA - GDH1 for a Co - IP assay .

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Figure 6C GDH1-K503 or GDH1-K527 acetylation occurred in both the cytosol and nucleus.D, The levels of both AcK503 and AcK527 were analysed when cells were transfected with small interfering RNA (siRNA) targeting TIP60, PCAF, CBP or p300 (D)F HCT116 and SW480 cells were transfected with or without FLAG-p300. The p300-associated proteins were enriched with M2-FLAG beads and subjected to immunoblot analysis using antibodies against GDH1, GDH1-K503 or GDH1-K527 acetylation.K The HIF1A gene was transiently knocked down, and HCT116 cells were cotransfected with FLAG-p300 and HA-GDH1 for a Co-IP assay.


Figure 7A , Cell viability in the indicated cell lines under hypoxia were analysed by trypan blue ( A ) ( n = 3 ) . Data information : Data are mean ± SD from the biological replicates ( A , Statistics : two - way ANOVA with Tukey ' s HSD post hoc test ( AI Detection of AcK503 / 527 - GDH1 expression in CRC grades I + Ⅱ and III + Ⅳ . ( grade I + II , n = 21 ; grade III + IV , n = 12 ) . J Detection of AcK503 / 527 - GDH1 expression in patients with ( ＋ ) or without ( － ) recurrence . ( － , n = 17 ; ＋ , n = 17 ) . Data information : Data are mean ± SD from the biological replicates I , J Statistics : Mann - Whitney U - test ( I , J ) K Patient survival corresponding to different levels of AcK503 / 527 - GDH1 expression ( High , n = 38 ; Low , n = 32 ) . Data information : Statistics : log - rank test ( K ) .

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 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

Figure 7A, Cell viability in the indicated cell lines under hypoxia were analysed by trypan blue (A) (n=3). Data information: Data are mean ± SD from the biological replicates (A, Statistics: two-way ANOVA with Tukey's HSD post hoc test (AI Detection of AcK503/527-GDH1 expression in CRC grades I+Ⅱ and III+Ⅳ. (grade I+II, n=21; grade III+IV, n=12). J Detection of AcK503/527-GDH1 expression in patients with (＋) or without (－) recurrence. (－, n=17; ＋, n=17). Data information: Data are mean ± SD from the biological replicates I, J Statistics: Mann-Whitney U-test (I, J)K Patient survival corresponding to different levels of AcK503/527-GDH1 expression (High, n=38; Low, n=32). Data information: Statistics: log-rank test (K).


Figure 1B , C Both mRNA and protein levels of RBM47 were elevated in SeV - infected ( B ) or IFN - α - stimulated ( C ) 293T , HFF , HUVEC , and THP - 1 cells . Cells were infected with SeV ( MOI = 1 ) or stimulated with IFN - α for 6 or 12 h , and the expression of RBM47 mRNA ( at 6 and 12 h post treatment ) and protein ( at 6 and 12 h post treatment ) were tested . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 1B, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 2C , D Levels of viral genome , protein , and progeny of VSV ( C ) or HSV - 1 ( D ) were quantified in virus - infected 293T cells with or without RBM47 ectopic expression at the indicated time points . The viral protein levels were tested 12 h post VSV infection and 24 h post HSV - 1 infection . The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 2C, D Levels of viral genome, protein, and progeny of VSV (C) or HSV-1 (D) were quantified in virus-infected 293T cells with or without RBM47 ectopic expression at the indicated time points. The viral protein levels were tested 12 h post VSV infection and 24 h post HSV-1 infection.The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments.**P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 3D , E mRNA expression of IFIT1 , ISG15 , and Cig5 in SeV - infected 293T cells overexpressing RBM47 ( D ) or RBM47 knocked down cells ( E ) . Cells were infected with SeV for 24 h , and gene expression was determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . F The expression of IFIT1 , ISG15 , and Cig5 in RBM47 - overexpressing or control 293T cells stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . G The expression of IFIT1 , ISG15 , and Cig5 in WT and RBM47 + / - peritoneal macrophages stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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 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 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

Figure 3D, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).F The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).G The expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 4B IFNAR1 mRNA stability after DRB treatment . 293T cells were transfected with RBM47 - Flag or empty vectors and treated with DRB ( 50 μg / mL ) . Total RNA was harvested at 0 , 15 , 30 , 45 , and 60 min , and IFNAR1 mRNA levels were measured using qRT - PCR and normalized to β - actin . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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Figure 4B IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 5A mRNA of IFNAR1 , but not IFNAR2 or STAT1 , was stabilized by RBM47 ectopic expression in 293T cells with or without IFN - α stimulation . Cells were treated with IFN - α or PBS for 6 h , and the relative gene expression levels were determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments . F , G Protein levels of IFNAR1 and phosphorylation levels of TYK2 , STAT1 , and STAT2 in RBM47 - overexpressing ( F ) or knockdown ( G ) 293T cells . Cells were treated with PBS or IFN - α for 6 h , and the relative band densities were analyzed using ImageJ software and normalized to GAPDH . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments .

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 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

Figure 5A mRNA of IFNAR1, but not IFNAR2 or STAT1, was stabilized by RBM47 ectopic expression in 293T cells with or without IFN-α stimulation. Cells were treated with IFN-α or PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.F, G Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


Figure 6G RBM47 + / + and RBM47 + / - mice were infected with HSV - 1 ( 1 × 107 pfu ) via caudal vein injection . Viral genome DNA and progeny titers were tested using blood samples from infected mice . Data are represented as the means ± SD of n = 6 biological replicates . H qRT - PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV - 1 - infected mice . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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Figure 6G RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 7D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient ( IFNAR1 - / - ) mice ( n = 6 per group ) infected with VSV . Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h . Each dot represents one mouse . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 7D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient (IFNAR1-/-) mice (n = 6 per group) infected with VSV. Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h. Each dot represents one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 1 ( B , C ) Functional classification and subcellular localization of identified lysine - acetylated proteins . Lysine - acetylated proteins identified over all experiments were classified according to MapMan categoriesOver - or underrepresentation of categories was determined using a Fisher ' s exact test with all proteins identified at 1 % FDR as background population . Blue and red arrows mark categories significantly enriched at 5 % FDR ( Benjamini - Hochberg ) and a 1 . 5 - fold - change cut - off .

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Figure 1(B, C) Functional classification and subcellular localization of identified lysine-acetylated proteins. Lysine-acetylated proteins identified over all experiments were classified according to MapMan categoriesOver- or underrepresentation of categories was determined using a Fisher's exact test with all proteins identified at 1% FDR as background population. Blue and red arrows mark categories significantly enriched at 5% FDR (Benjamini-Hochberg) and a 1.5-fold-change cut-off.


Figure 3Figure 3 . Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin ( A , C ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict lysine acetylation site ratios ( A , B ) for inhibitor treatment vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization according to SUBA4 database . Blue , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing line indicates that the significance threshold was not reached by any of the data points . Figure 3 . Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A ( B , D ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict lysine acetylation site ratios ( A , B ) for inhibitor treatment vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization according to SUBA4 database . Blue , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing line indicates that the significance threshold was not reached by any of the data points . Figure 3 . Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin ( A , C ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict protein ratios ( C , D ) for inhibitor treatment vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization according to SUBA4 database . Blue , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing line indicates that the significance threshold was not reached by any of the data points . cancel Figure 3 . Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A ( B , D ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict protein ratios ( C , D ) for inhibitor treatment vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization according to SUBA4 database . Blue , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing line indicates that the significance threshold was not reached by any of the data points .

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 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 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

Figure 3Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C)versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points. cancel Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.


Figure 4Figure 4 . HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis , its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors . ( A ) GFP localization ( green ) of the HDA14 - GFP fusion construct in Arabidopsis protoplasts ( 35S : HDA14 : GFP ) from stable transformants . The mitochondrial marker TMRM is depicted in purple . GFP + TMRM shows the overlay image of 35S : HDA14 : GFP and TMRM , AF indicates the chlorophyll autofluorescence and BF the ( B ) Deacetylase activity of the recombinant 6xHis - HDA14 protein using a colorimetric assay . Co2 + and Zn2 + were used as cofactors , apicidin ( 100 µM ) and trichostatin A ( 5 µM ) as deacetylase - inhibitors ( n = 5 , ± SD ) .

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Figure 4Figure 4. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors. (A) GFP localization (green) of the HDA14-GFP fusion construct in Arabidopsis protoplasts (35S:HDA14:GFP) from stable transformants. The mitochondrial marker TMRM is depicted in purple. GFP+TMRM shows the overlay image of 35S:HDA14:GFP and TMRM, AF indicates the chlorophyll autofluorescence and BF the(B) Deacetylase activity of the recombinant 6xHis-HDA14 protein using a colorimetric assay. Co2+ and Zn2+ were used as cofactors, apicidin (100 µM) and trichostatin A (5 µM) as deacetylase-inhibitors (n = 5, ±SD).


Figure 5Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves under growth light ( A , D ) Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points . cancel Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves in isolated thylakoids ( B , E ) Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points . Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves under low light conditions ( C , F ) . Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points . Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves under growth light ( A , D ) Volcano plots depict protein ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points . Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves in isolated thylakoids ( B , E ) Volcano plots depict protein ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points . Figure 5 . Differential lysine acetylation and protein expression in hda14 vs . wild - type leaves under low light conditions ( C , F ) . Volcano plots depict protein ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using the LIMMA package . Orange , protein with nuclear localization ; Green , protein with plastidial localization ; purple triangles , proteins of the Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , proteins of the light reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , proteins with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR line indicates that the 5 % threshold was not reached by any of the data points .

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 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 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 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 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 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

Figure 5Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under growth light (A, D)Volcano plots depict lysine acetylation site ratios (A-C, top row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points. cancel Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves in isolated thylakoids (B, E)Volcano plots depict lysine acetylation site ratios (A-C, top row)for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under low light conditions (C,F). Volcano plots depict lysine acetylation site ratios (A-C, top row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under growth light (A, D) Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves in isolated thylakoids (B, E) Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.Figure 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under low light conditions (C,F). Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 6Figure 6 . RuBisCO activity and RuBisCO activation state is increased in the hda14 mutant under low light conditions . ( A ) RuBisCO initial and total activity in WT and hda14 in low light treated plants . Initial activity was measured directly upon extraction . For the total activity samples were incubated with H2CO3 for 3 minutes to fully carbamylate the active site of RuBisCO ( n = 10 , * t - test p - value < 0 . 05 ) . ( B ) RuBisCO activation state . ( C ) ATPase activity of recombinant 6x - HisRCAß1 WT , K438Q and K438R with ATP and ADP / ATP = 0 . 11 , respectively ( n = 3 , * t - test p - value < 0 . 05 , + t - test p - value < 0 . 1 ) .

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 B-PANEL_START 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

Figure 6Figure 6. RuBisCO activity and RuBisCO activation state is increased in the hda14 mutant under low light conditions. (A) RuBisCO initial and total activity in WT and hda14 in low light treated plants. Initial activity was measured directly upon extraction. For the total activity samples were incubated with H2CO3 for 3 minutes to fully carbamylate the active site of RuBisCO (n = 10, *t-test p-value < 0.05).(B) RuBisCO activation state.(C) ATPase activity of recombinant 6x-HisRCAß1 WT, K438Q and K438R with ATP and ADP/ATP= 0.11, respectively (n = 3, *t-test p-value < 0.05, +t-test p-value< 0.1).


Figure 1A Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1 - TAP followed by label - free quantification ( LFQ ) using liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) . Proteins above the curved lines show a statistically significant enrichment according to the t test value . B , C Sucrose density gradient UV - profile after in vitro splitting assays ( B ) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD . ( C ) ; SF = splitting factors including Dom34 , Hbs1 , ABCE1 and eIF6 ; ( + ) = 4 - fold molar excess of eIF3j ; ( + + ) = 20 - fold molar excess of eIF3j .

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 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

Figure 1A Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1-TAP followed by label-free quantification (LFQ) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins above the curved lines show a statistically significant enrichment according to the t test value.B, C Sucrose density gradient UV-profile after in vitro splitting assays (B) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD. (C); SF = splitting factors including Dom34, Hbs1, ABCE1 and eIF6; (+) = 4-fold molar excess of eIF3j; (++) = 20-fold molar excess of eIF3j.


Figure 2SARSCoV - 2 MA30 infection in C57BL / 6 mice 7 days follow up . PEGSerp - 1 treatment significantly improved Mean ( C ) weights ( N = 20 mice , C mean ± SD ) ) Data analysis information : Mean ± SD ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Unpaired T tests ; indicated by line at top with subgroup analyses in brackets below ; Prophylactic treatment black - Saline , red - PEGSerp - 1 treatments . E Micrographs illustrate lung consolidation in Saline and PEGSerp - 1 treated lungs . Large blue arrows indicate areas of consolidation in H & E stained lung sections ( Mag 2X ) . G , Combined weights for the 4 and 7 day follow up studies demonstrate consistently improved mean weights ( G ; p < 0 . 0153 - 0 . 0001 ) with PEGSerp - 1 treatments in MA30 infected mice . ( N = 39mice , mean ± SD ) Data analysis information : black - Saline , red - PEGSerp - 1 treatments . I Micrographs illustrate lung consolidation divided by total section analyzed in Saline and PEGSerp - 1 treated MA30 infected mice at 4 days follow up . PEGSerp - 1 delayed treatment at 10ng / gm starting 2 days after infection reduced weight loss ( L ; p < 0 . 001 - 0 . 05 ) . ( N = 24 mice , mean ± SE ) Data analysis information : Mean ± SE ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; blue - Saline , green and blue - PEGSerp - 1 .

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 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 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 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

Figure 2SARSCoV-2 MA30 infection in C57BL/6 mice 7 days follow up. PEGSerp-1 treatment significantly improved Mean (C) weights (N = 20 mice, C mean ± SD)) Data analysis information: Mean ± SD ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Unpaired T tests; indicated by line at top with subgroup analyses in brackets below; Prophylactic treatment black - Saline, red -PEGSerp-1 treatments.E Micrographs illustrate lung consolidation in Saline and PEGSerp-1 treated lungs. Large blue arrows indicate areas of consolidation in H&E stained lung sections (Mag 2X).G, Combined weights for the 4 and 7 day follow up studies demonstrate consistently improved mean weights (G; p < 0.0153- 0.0001) with PEGSerp-1 treatments in MA30 infected mice. (N = 39mice, mean ±SD) Data analysis information: black - Saline, red -PEGSerp-1 treatments.I Micrographs illustrate lung consolidation divided by total section analyzed in Saline and PEGSerp-1 treated MA30 infected mice at 4 days follow up.PEGSerp-1 delayed treatment at 10ng/gm starting 2 days after infection reduced weight loss (L; p < 0.001-0.05). (N = 24 mice, mean ± SE) Data analysis information: Mean ± SE ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; blue - Saline, green and blue - PEGSerp-1.


Figure 3A , B Micrographs illustrating iNOS positive M1 macrophage cell infiltrates on IHC stained lung sections in Saline and PEGSerp - 1 ( A ) treated mice with significantly reduced IHC positive cell counts at 4 days follow up after PEGSerp - 1 prophylactic treatment ( B ; * p < 0 . 045 ) . IHC stained lung sections ( Mag 40X ) . Data information : Mean ± S . E . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; back circles Saline , red circles PEGSerp - 1 treatments . C , D iNOS positive infiltrates in IHC stained lung sections with Saline or PEGSerp - 1 ( C ) treatment with significantly reduced IHC positive cell counts at 7 days follow up with prophylactic PEGSerp - 1 treatment ( D ; * * p < 0 . 0052 ) . IHC stained lung sections ( Mag 40X ) . Data information : Mean ± S . E . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; back circles Saline , red circles PEGSerp - 1 treatments . E Delayed PEGSerp - 1 10ng / gm dose treatment given 2 days after infection also reduced iNOS positive cell counts ( * p < 0 . 0368 ) . Data information : Mean ± S . E . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; blue circles Saline , red and green circles PEGSerp - 1 treatments . G CD3 + T cell counts were not significantly altered at 4 days ( p = 0 . 1058 ) . H CD4 + T cell counts are not reduced at 4 days ( p = 0 . 5031 ) but are reduced at 7 days ( * p < 0 . 0454 ) with PEGSerp - 1 treatments . I CD8 positive T cell counts on IHC stained sections have a trend toward an increase at 4 days ( p = 0 . 1503 ) and a significant reduction at 7 days ( * p < 0 . 0303 ) with PEG Serp - 1 treatments . J IHC for Neutrophil marker Ly6G did not detect significant changes in PEGSerp - 1 treatment at days 4 ( p = 0 . 0860 ) and 7 ( p = 0 . 6570 ) . Data information : Mean ± S . E . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; back and blue circles Saline , red and green circles PEGSerp - 1 treatments .

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 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 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 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

Figure 3A, B Micrographs illustrating iNOS positive M1 macrophage cell infiltrates on IHC stained lung sections in Saline and PEGSerp-1 (A) treated mice with significantly reduced IHC positive cell counts at 4 days follow up after PEGSerp-1 prophylactic treatment (B; *p < 0.045). IHC stained lung sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.C, D iNOS positive infiltrates in IHC stained lung sections with Saline or PEGSerp-1 (C) treatment with significantly reduced IHC positive cell counts at 7 days follow up with prophylactic PEGSerp-1 treatment (D; **p < 0.0052). IHC stained lung sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.E Delayed PEGSerp-1 10ng/gm dose treatment given 2 days after infection also reduced iNOS positive cell counts (*p < 0.0368). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; blue circles Saline, red and green circles PEGSerp-1 treatments.G CD3 + T cell counts were not significantly altered at 4 days (p = 0.1058). H CD4 + T cell counts are not reduced at 4 days (p =0.5031) but are reduced at 7 days (*p < 0.0454) with PEGSerp-1 treatments. I CD8 positive T cell counts on IHC stained sections have a trend toward an increase at 4 days (p = 0.1503) and a significant reduction at 7 days (*p < 0.0303) with PEG Serp-1 treatments. J IHC for Neutrophil marker Ly6G did not detect significant changes in PEGSerp-1 treatment at days 4 (p = 0.0860) and 7 (p = 0.6570). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments.


Figure 4iNOS M1 stained macrophage invasion in Saline control treated mice ( A ) Black arrows point to brown iNOS positive macrophage on IHC stained lung sections ( Mag 40X ) . PEGSerp - 1 treatment reduced iNOS + macrophage infiltration . iNOS M1 stained macrophage invasion is significantly reduced with PEGSerp - 1 treatment ( B ) . Black arrows point to brown iNOS positive macrophage on IHC stained lung sections ( Mag 40X ) . H & E stained histology sections of lung illustrating consolidation in Saline treated mice ( E ) saline treated uninfected mouse tissues had similar levels of consolidation Black arrows point to brown iNOS positive macrophage on IHC stained lung sections ( Mag 40X ) . PEGSerp - 1 treatment reduced lung consolidation H & E stained histology sections of lung illustrating consolidation is reduced by PEGSerp - 1 treatment ( F ) . Black arrows point to brown iNOS positive macrophage on IHC stained lung sections ( Mag 40X ) .

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 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 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 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

Figure 4iNOS M1 stained macrophage invasion in Saline control treated mice (A) Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).PEGSerp-1 treatment reduced iNOS + macrophage infiltration. iNOS M1 stained macrophage invasion is significantly reduced with PEGSerp-1 treatment (B). Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).H & E stained histology sections of lung illustrating consolidation in Saline treated mice (E) saline treated uninfected mouse tissues had similar levels of consolidation Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).PEGSerp-1 treatment reduced lung consolidation H & E stained histology sections of lung illustrating consolidation is reduced by PEGSerp-1 treatment (F). Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).


Figure 5A , B Micrographs illustrating iNOS positive cells in Saline ( A ) and PEGSerp - 1 ( B ) treated mice at 4 days follow up . IHC stained sections 40X .

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Figure 5A, B Micrographs illustrating iNOS positive cells in Saline (A) and PEGSerp-1 (B) treated mice at 4 days follow up. IHC stained sections 40X.


Figure 6PEGSerp - 1 treated mouse lung sections from MA10 at day 2 follow up have significant reduction in uPAR positive stained areas ( A ) with reduced positively stained hemorrhagic areas in lungs at 2 days in MA10 infected BALB / c ( B ; p < 0 . 041 ) and reduced uPAR + stained cell counts at 4 days ( C ; p < 0 . 0202 ) and 7 days ( C in alveoli ( p < 0 . 0148 ) in MA30 infected C57BL / 6 mice . With delayed treatments uPAR is reduced at 7 days follow up ( D ; ANOVA p < 0 . 0001 , p < 0 . 0002 for PEGSerp - 1 at 10µg / kg ) . FX staining was reduced with prophylactic PEGSerp - 1 at day 7 but not at day 4 follow up ( E ; ANOVA p < 0 . 0008 , p < 0 . 0001 for day 7 and p = 0 . 5148 for day 4 ) . Data information : Mean ± SE . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; back and blue circles Saline , red and green circles PEGSerp - 1 treatmentsC5b - 9 staining is reduced in prophylactic PEGSerp - 1 treatment in MA30 infected C57BL / 6 mice on IHC stained sections ( H ) with significant reductions at day 4 ( I ; p < 0 . 0114 ) , and a nonsignificant trend at day 7 follow up ( p = 0 . 5694 ) . With delayed PEGSerp - 1 treatment C5b / 9 detection on IHC is significantly reduced by both PEGSerp - 1 doses ( J ; p < 0 . 0131 ) . Data information : Mean ± SE . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below ; back and blue circles Saline , red and green circles PEGSerp - 1

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 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

Figure 6PEGSerp-1 treated mouse lung sections from MA10 at day 2 follow up have significant reduction in uPAR positive stained areas (A) with reduced positively stained hemorrhagic areas in lungs at 2 days in MA10 infected BALB/c (B; p < 0.041) and reduced uPAR + stained cell counts at 4 days (C; p < 0.0202) and 7 days (C in alveoli (p < 0.0148) in MA30 infected C57BL/6 mice. With delayed treatments uPAR is reduced at 7 days follow up (D; ANOVA p< 0.0001, p < 0.0002 for PEGSerp-1 at 10µg/kg). FX staining was reduced with prophylactic PEGSerp-1 at day 7 but not at day 4 follow up (E; ANOVA p < 0.0008, p < 0.0001 for day 7 and p = 0.5148 for day 4). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatmentsC5b-9 staining is reduced in prophylactic PEGSerp-1 treatment in MA30 infected C57BL/6 mice on IHC stained sections (H) with significant reductions at day 4 (I; p <0.0114), and a nonsignificant trend at day 7 follow up (p = 0.5694). With delayed PEGSerp-1 treatment C5b/9 detection on IHC is significantly reduced by both PEGSerp-1 doses (J; p < 0.0131). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments


Figure 7D - G In MA30 infected C57BL / 6 mice increased S protein is seen on IHC staining seen in lung sections from Saline treated mice ( D ) is reduced with PEGSerp - 1 treatment at 7 days follow up ( E , F ; p < 0 . 001 ) and in the small vessels ( G ; p < 0 . 0364 ) . Data information : Mean ± SE . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; ANOVA indicated by line at top with subgroup analyses in brackets below .

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Figure 7D-G In MA30 infected C57BL/6 mice increased S protein is seen on IHC staining seen in lung sections from Saline treated mice (D) is reduced with PEGSerp-1 treatment at 7 days follow up (E, F; p < 0.001) and in the small vessels (G; p < 0.0364). Data information: Mean ±SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below.


Figure 8A , B Relative gene expression is significantly increased for SARS - CoV - 2 MA30 viral E gene ( A ; p < 0 . 0017 ) and interferon response ISG15 ( B ; p < 0 . 0044 ) in Saline treated infected lung extracts at 4 days follow up , when compared to uninfected mouse lungs . Data information : Gene expression is normalized to GAPDH expression . Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control . ( ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) . Relative changes in gene expression for SARS - CoV - 2 infections and associated modulation in inflammatory and coagulation responses are increased after SARS - CoV - 2 MA30 infection in lung and heart tissues ( SARSCoV - 2 MA30 infection - N = 18 mice 4 days follow up ; N = 20 mice 7 days follow up ; Uninfected mice - N = 2 ) . PEGSerp - 1 treatment significantly modified gene expression for uPAR and complement pathways . C - L Inflammatory cytokines and growth factors are significantly increased with infection ; IL - 1 ( C ; p < 0 . 0003 ) , IL - 6 ( D p < 0 . 0001 ) , IL - 10 ( E ; p < 0 . 0001 ) ) , TNF ( F ; p < 0 . 0044 ) , TGFb ( G ; p < 0 . 0022 ) are increased with SARS - CoV - 2 infection at 4 days follow up ; with VEGF the exception ( H ; p = 0 . 4695 ) Factors in the coagulation pathway , fX ( I ; p < 0 . 0001 ) , tPA ( J ; p < 0 . 0382 ) , and neuroserpin ( K ; p = 0 . 1883 ) are also increased . None of these markers are modified by PEGSerp - 1 treatments . Data information : Gene expression is normalized to GAPDH expression . Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control . ( ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) .

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 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

Figure 8A, B Relative gene expression is significantly increased for SARS-CoV-2 MA30 viral E gene (A; p < 0.0017) and interferon response ISG15 (B; p < 0.0044) in Saline treated infected lung extracts at 4 days follow up, when compared to uninfected mouse lungs. Data information: Gene expression is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in inflammatory and coagulation responses are increased after SARS-CoV-2 MA30 infection in lung and heart tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for uPAR and complement pathways. C-L Inflammatory cytokines and growth factors are significantly increased with infection; IL-1 (C; p < 0.0003), IL-6 (D p < 0.0001), IL-10 (E; p < 0.0001) ), TNF (F; p < 0.0044), TGFb (G; p < 0.0022) are increased with SARS-CoV-2 infection at 4 days follow up; with VEGF the exception (H; p = 0.4695 ) Factors in the coagulation pathway, fX (I; p < 0.0001), tPA (J; p < 0.0382), and neuroserpin (K; p = 0.1883 ) are also increased. None of these markers are modified by PEGSerp-1 treatments. Data information: Gene expression is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).


Figure 9Relative changes in gene expression for SARS - CoV - 2 infections and associated modulation in coagulation and immune responses are increased after SARS - CoV - 2 MA30 infection in lung tissues ( SARSCoV - 2 MA30 infection - N = 18 mice 4 days follow up ; N = 20 mice 7 days follow up ; Uninfected mice - N = 2 ) . PEGSerp - 1 treatment significantly modified gene expression for uPAR A - C Gene expression for PEGserp - 1 targets are increased after infection and modified by PEGSerp - 1 treatments . uPA ( A ; p = 0 . 3263 ) , PAI - 1 ( B ; p < 0 . 0001 ) and uPAR ( C ; p < 0 . 0001 ) were increased in infected mouse lung extracts at 4 days follow up when compared to uninfected lungs with SARS - CoV - 2 MA30 infection . PEG Serp - 1 treatment produced a significant increase in uPAR ( C ; p < 0 . 0157 ) . Data information : N = 19 mice at 4 days follow up ; N = 20 mice at 7 days follow up . ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) . Relative changes in gene expression for SARS - CoV - 2 infections and associated modulation in coagulation and immune responses are increased after SARS - CoV - 2 MA30 infection in lung tissues ( SARSCoV - 2 MA30 infection - N = 18 mice 4 days follow up ; N = 20 mice 7 days follow up ; Uninfected mice - N = 2 ) . PEGSerp - 1 treatment significantly modified gene expression for complement pathways . D - F Complement pathways genes encoding C3 ( D ; p < 0 . 0021 ) , C5 ( E ; p < 0 . 001 ) and C1INh ( F ; p < 0 . 0008 ) were also significantly increased with infection . PEG Serp - 1 treatment produced a borderline decrease in C5 ( E ; p = 0 . 0623 ) and a significant increase in C1Inh ( F ; p < 0 . 0309 ) . Data information : N = 19 mice at 4 days follow up ; N = 20 mice at 7 days follow up . ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) . Relative changes in gene expression for SARS - CoV - 2 infections and associated modulation in coagulation and immune responses are increased after SARS - CoV - 2 MA30 infection in lung and heart tissues ( SARSCoV - 2 MA30 infection - N = 18 mice 4 days follow up ; N = 20 mice 7 days follow up ; Uninfected mice - N = 2 ) . PEGSerp - 1 treatment significantly modified gene expression for complement pathways . G - I At 7 days follow up , relative gene expression is no longer increased for C1Inh with PEGSerp - 1 treatment in MA30 infected mouse lungs ( G ; p = 0 . 5956 ) , but C1Inh was significantly increased in myocardial extracts with PEGSerp - 1 treatment at 7 days ( H ; p < 0 . 0419 ) . C5 was not significantly increased with infection ( I ; p = 0 . 1601 ) Data information : N = 19 mice at 4 days follow up ; N = 20 mice at 7 days follow up . ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) . Relative changes in gene expression for SARS - CoV - 2 infections and associated modulation in coagulation and immune responses are increased after SARS - CoV - 2 MA30 infection in lung and heart tissues ( SARSCoV - 2 MA30 infection - N = 18 mice 4 days follow up ; N = 20 mice 7 days follow up ; Uninfected mice - N = 2 ) . PEGSerp - 1 treatment significantly modified gene expression for uPAR J - L PAI - 1 gene expression was not increased in lung extracts with PEGSerp - 1 treatment at 7 days follow up in MA30 infected mouse lungs ( J ; p = 0 . 2403 ) , but PAI - 1 expression was significantly increased at 7 days follow up in heart extracts ( K ; p < 0 . 0454 ) . uPAR was not significantly increased in lung extracts at 7 days follow up ( p = 0 . 1514 ) . A time course for gene expression illustrates a peak time for changes in C1Inh gene expression demonstrates greater changes at 4 days when compared to day2 and day15 ( L ) . Data information : N = 19 mice at 4 days follow up ; N = 20 mice at 7 days follow up . ANOVA indicated by line at top with subgroup analyses below ; infected mice - blue circles Saline , red circle PEGSerp - 1 treatments , green circle uninfected mice ) .

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 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 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 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

Figure 9Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2 MA30 infection in lung tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for uPAR A-C Gene expression for PEGserp-1 targets are increased after infection and modified by PEGSerp-1 treatments. uPA (A; p = 0.3263), PAI-1 (B; p < 0.0001) and uPAR (C; p < 0.0001) were increased in infected mouse lung extracts at 4 days follow up when compared to uninfected lungs with SARS-CoV-2 MA30 infection. PEG Serp-1 treatment produced a significant increase in uPAR (C; p < 0.0157). Data information: N = 19 mice at 4 days follow up; N = 20 mice at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2 MA30 infection in lung tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for complement pathways. D-F Complement pathways genes encoding C3 (D; p < 0.0021), C5 (E; p < 0.001) and C1INh (F; p < 0.0008) were also significantly increased with infection. PEG Serp-1 treatment produced a borderline decrease in C5 (E; p = 0.0623) and a significant increase in C1Inh (F; p < 0.0309). Data information: N = 19 mice at 4 days follow up; N = 20 mice at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2 MA30 infection in lung and heart tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for complement pathways. G-I At 7 days follow up, relative gene expression is no longer increased for C1Inh with PEGSerp-1 treatment in MA30 infected mouse lungs (G; p = 0.5956), but C1Inh was significantly increased in myocardial extracts with PEGSerp-1 treatment at 7 days (H; p < 0.0419). C5 was not significantly increased with infection (I; p = 0.1601) Data information: N = 19 mice at 4 days follow up; N = 20 mice at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2 MA30 infection in lung and heart tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for uPAR J-L PAI-1 gene expression was not increased in lung extracts with PEGSerp-1 treatment at 7 days follow up in MA30 infected mouse lungs (J; p = 0.2403), but PAI-1 expression was significantly increased at 7 days follow up in heart extracts (K; p < 0.0454). uPAR was not significantly increased in lung extracts at 7 days follow up (p = 0.1514). A time course for gene expression illustrates a peak time for changes in C1Inh gene expression demonstrates greater changes at 4 days when compared to day2 and day15 (L). Data information: N = 19 mice at 4 days follow up; N = 20 mice at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).