Figure 6 ( G ) Human choroidal EC ( HCEC ) and mouse retinal EC ( MREC ) were treated with LIF 10 ng , LIF 100 ng for 30 min . ( PBS was used as control ) . STAT3 phosphorylation was detected by western blot . ( H ) H & E staining of LIFR and CD31 in mouse eye sections . The magnified images of the dashed line boxes , a and b , are shown on the right panel . Box a shows LIFR staining in retina and box b showed LIFR staining in RPE / choroid layer . ( I ) Whole mount staining of LIFR and CD31 in mouse retina . The fluorescent staining for CD31 ( green ) and LIFR ( red ) is shown . White arrows indicate the co - staining of CD31 and LIFR on retinal ECs .

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Figure 6(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.(H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.(I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.


Figure 7Violin plots of the log scale normalized expression of LIF receptor subunits , IL6ST ( A ) , LIFR ( B ) , LIF ( C ) , IL6R ( D ) in all cell types in the human choroid Note the wide distribution of IL6ST ( gp130 ) , the common subunit to all receptors of the IL - 6 family ( A ) . In contrast , LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes ( B ) . LIF is expressed in fibroblasts , macrophages / monocytes and pericytes and minimally in ECs ( C ) . The specific IL - 6 receptor ( IL6R ) is highly expressed in macrophage / monocytes ( D ) , while LIFR has little expression in these cells ( B ) . Violin plots of the log scale normalized expression of LIF receptor subunits KDR ( E ) in all cell types in the human choroid KDR shows , as expected , high expression selectively in EC ( E ) .

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Figure 7Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) in all cell types in the human choroid Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B).Violin plots of the log scale normalized expression of LIF receptor subunits KDR (E) in all cell types in the human choroid KDR shows, as expected, high expression selectively in EC (E).


Figure 2A . Total lipids were extracted from PfCDPK7 - KO or 3D7 parasites and separated by HPTLC . After quantification by GC - MS content relative to total fatty acids was determined . Relative abundance of individual phospholipids was quantified . Disruption of PfCDPK7 caused a significant decrease in PC ( mean ± SEM , n = 3 ; biological replicates , * * * p < 0 . 001 , t - test ) . E . 3D7 or PfCDPK7 - KO parasites were cultured in the presence or absence of 200 μM choline in culture medium . After 3 days , parasitemia was determined by counting parasites on thin blood smears ( mean ± SEM , n = 6 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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Figure 2A. Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual phospholipids was quantified. Disruption of PfCDPK7 caused a significant decrease in PC (mean± SEM, n=3; biological replicates, ***p<0.001, t-test).E. 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days, parasitemia was determined by counting parasites on thin blood smears (mean± SEM, n=6; biological replicates, *p< 0.05, ***p<0.001, ANOVA).


Figure 3A - B . 3D7 ( A ) or parasites overexpressing EK - GFP ( B ) were used to prepare protein lysates followed by immunoprecipitation using anti - PfCDPK7 or anti - GFP antibody , respectively . Subsequently , protein lysate or indicated IP were subjected to Western blotting using anti - PMT ( A ) , anti - GFP ( B ) . PMT was found to co - IP with PfCDPK7 ( A , lane 2 ) and EK - GFP with PfCDPK7 ( B , lane 3 ) . C . 3D7 and PfCDPK7 - KO parasites at schizont stage ( ~ 40 h ) were used for Western blotting using anti - PMT antisera . Actin served as a loading control . Right panel : Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin . Fold change in PMT was determined with respect to 3D7 ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , t - test ) .

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Figure 3A-B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3).C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean± SEM, n=4; biological replicates, **p<0.01, t-test).


Figure 4A . Parasites overexpressing EK - GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first ( 48 h . p . i ) and second cycle ( 96 h . p . i ) . Fold change in parasitemia of EK _ S37A parasites with respect to WT EK overexpressing parasites was compared , which was significantly reduced ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , ANOVA ) . C . IFA was performed on parasites expressing WT EK - GFP or its S37A mutant 6 - 12h post - invasion to detect ( i ) RAP1 , which is transferred to PV post invasion , and ( ii ) MSP1 which is located at the PVM . Parasite expressing WT EK - GFP exhibited typical round ring like shape . In contrast , several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV ( Scale bar - 1 μm ) . E . Parasites EK - GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts / segmentors . The graph shows average number of merozoites per schizont from three biological replicates . Each replicate represents an average of merozoites from 30 - 40 schizonts . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ) .

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Figure 4A. Parasites overexpressing EK-GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first (48 h.p.i) and second cycle (96 h.p.i). Fold change in parasitemia of EK_S37A parasites with respect to WT EK overexpressing parasites was compared, which was significantly reduced (mean± SEM, n=3; biological replicates, *p<0.05, ANOVA).C. IFA was performed on parasites expressing WT EK-GFP or its S37A mutant 6-12h post-invasion to detect (i) RAP1, which is transferred to PV post invasion, and (ii) MSP1 which is located at the PVM. Parasite expressing WT EK-GFP exhibited typical round ring like shape. In contrast, several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV (Scale bar-1 μm).E. Parasites EK-GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts/segmentors. The graph shows average number of merozoites per schizont from three biological replicates. Each replicate represents an average of merozoites from 30-40 schizonts. (mean± SEM, n=3; biological replicates, *p<0.05, t-test).


Figure 5A . 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h . Subsequently , parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7 . While PfCDPK7 was present in puncta proximal to ER ( left panel ) , inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme ( right panel ) ( Scale bar - 1 μm ) . C . 3D7 parasites were treated with DMSO or 50nM MMV048 for 20 - 24h . Subsequently , IFA was performed ~ 24h post - invasion to detect RAP1 and MSP1 ( left panel ) Right panel , Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed ( mean ± SEM , n = 3 ; biological replicates , * * p < 0 . 01 , t - test ; Scale bar - 1 μm ) .

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Figure 5A. 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h. Subsequently, parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7. While PfCDPK7 was present in puncta proximal to ER (left panel), inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme (right panel) (Scale bar-1 μm).C. 3D7 parasites were treated with DMSO or 50nM MMV048 for 20-24h. Subsequently, IFA was performed ~24h post-invasion to detect RAP1 and MSP1 (left panel) Right panel, Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed (mean± SEM, n=3; biological replicates, **p<0.01, t-test; Scale bar-1 μm).


Figure 6A - B . 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10 - 12h . Subsequently , parasites were incubated with 14C - Cho ( A ) or 14C - Eth ( B ) and were harvested after 12h . Total lipids were extracted and separated by TLC . The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry ( upper panel ) . Bottom Panel ( A and B ) , fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ; ns , not significant ) . C - D . UCT943 and MMV048 treated 3D7 ( C ) or EK - GFP overexpressing parasites ( D ) were subjected to Western blotting using anti - PMT ( C ) and anti - GFP ( D ) antibodies ( left panels ) . Right Panels : densitometry was performed for bands corresponding to PMT ( C ) or EK - GFP ( D ) , which were normalized with respect to Actin . Fold change in PMT or EK was determined with respect to DMSO treated parasites ( Mean ± SEM , n = 4 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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Figure 6A-B. 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10-12h. Subsequently, parasites were incubated with 14C-Cho (A) or 14C-Eth (B) and were harvested after 12h. Total lipids were extracted and separated by TLC. The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry (upper panel). Bottom Panel (A and B), fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined (mean±SEM, n=4; biological replicates, **p<0.01, ***p<0.001, ANOVA; ns, not significant).C-D. UCT943 and MMV048 treated 3D7 (C) or EK-GFP overexpressing parasites (D) were subjected to Western blotting using anti-PMT (C) and anti-GFP (D) antibodies (left panels). Right Panels: densitometry was performed for bands corresponding to PMT (C) or EK-GFP (D), which were normalized with respect to Actin. Fold change in PMT or EK was determined with respect to DMSO treated parasites (Mean± SEM, n=4; biological replicates, *p<0.05, ***p<0.001, ANOVA).


Figure 7B . Ring - infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4 - 64 and DAPI to assess lipid uptake . Subsequently , FM4 - 64 associated fluorescence was detected by fluorescence microscopy ( upper panel ) or quantitated by FACS ( lower panel ) and % change in FM4 - 64 uptake in inhibitor - treated parasites was determined . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) . D . Trophozoite stage infected erythrocytes were incubated with FM4 - 64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7 - KO parasites . Subsequently , FM4 - 64 associated fluorescence was visualized by live microscopy ( top panel ) and also analyzed by FACS and % change in FM4 - 64 uptake in KO parasites was determined ( bottom panel ) ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) .

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Figure 7B. Ring-infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4-64 and DAPI to assess lipid uptake. Subsequently, FM4-64 associated fluorescence was detected by fluorescence microscopy (upper panel) or quantitated by FACS (lower panel) and % change in FM4-64 uptake in inhibitor-treated parasites was determined. (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).D. Trophozoite stage infected erythrocytes were incubated with FM4-64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7-KO parasites. Subsequently, FM4-64 associated fluorescence was visualized by live microscopy (top panel) and also analyzed by FACS and % change in FM4-64 uptake in KO parasites was determined (bottom panel) (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).


Figure 1 ( C - E ) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate ( TCL ) and further increases by IFN - γ activation ( C , E ) . Equal protein amounts were loaded for phagosomal and TCL samples . Vimentin serves as loading control ; EEA1 as purity marker . ( D ) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome ( 30 min pulse , 0 , 30 , and 180 min chase , respectively ) . LAMP1 serves as purity marker . Representative images of three replicates . ( H ) Ubiquitin Chain Restriction ( UbiCRest ) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs . UT = untreated .

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Figure 1(C-E) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate (TCL) and further increases by IFN-γ activation (C, E). Equal protein amounts were loaded for phagosomal and TCL samples. Vimentin serves as loading control; EEA1 as purity marker. (D) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome (30 min pulse, 0, 30, and 180 min chase, respectively). LAMP1 serves as purity marker. Representative images of three replicates.(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.


Figure 3 ( B ) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264 . 7 cells and its increased abundance in response to IFN - γ activation . Rab7 serves as purity control , iNOS as activation control and vimentin as loading control . Representative image of three replicates . Relative mobilities of reference proteins ( masses in kDa ) are shown on the right of each blot .

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Figure 3(B) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264.7 cells and its increased abundance in response to IFN-γ activation. Rab7 serves as purity control, iNOS as activation control and vimentin as loading control. Representative image of three replicates. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.


Figure 4 ( A - B ) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells . ( C ) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes , shows that RNF115 ligase activity is required for the increase of phagosome maturation .

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Figure 4(A-B) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells. (C) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes, shows that RNF115 ligase activity is required for the increase of phagosome maturation.


Figure 5 ( C ) Immunofluorescence micrographs of VAMP8 ( green ) and Syntaxin - 7 ( STX7 ; red ) show strong colocation around 3 µm silica bead phagosomes ( white ) in BMA macrophages . Nuclei are stained with Dapi . Scale bar is 5 µm . To account for people with red - green colour - blindness , ( D ) Colocation of VAMP8 ( green ) and STX7 ( red ) on individual phagosomes between WT and RNF115 KO cells represented by intersection , Manders ' M1 and overlap coefficients . Data is represented as a box and whisker plot of total values across biological duplicate experiments , where the whiskers represent the minimum to maximum values and the central band indicates the median .

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Figure 5(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, (D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median.


Figure 7 ( D ) Box and whisker plot represents the quantification of the percentage ( % ) of the hepatic lesion area in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Haematoxylin and eosin ( H & E ) - stained liver sections of one representative animal per genotype are shown on the right , dotted line depicts the damaged area . ( E - F ) Box and whisker plots represent the average cell counts per high power field ( HPF ) of CD3 positive T - cells ( E ) , and CD68 positive macrophages ( F ) in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Representative immunohistochemistry sections are shown on the right .

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Figure 7(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area.(E-F) Box and whisker plots represent the average cell counts per high power field (HPF) of CD3 positive T-cells (E), and CD68 positive macrophages (F) in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Representative immunohistochemistry sections are shown on the right.


Figure 1A COS ‐ 1 cells co ‐ transfected with GFP ‐ ATP8A1 and myc ‐ CDC50A were stained for myc ‐ tag and TfnR , TGN46 , GM130 , VPS26 , CD63 , or LAMP2 . mCherry ‐ Rab11 was expressed in a cell in the top row . Nuclei were stained with DAPI ( in blue ) . Pearson ' s coefficient between GFP ‐ ATP8A1 and each organelle marker is shown in the merged image ( mean ± SD , n > 20 cells from two experiments ) . Scale bars , 10 μm . B COS ‐ 1 cells were treated with ATP8A1 siRNAs for 72 h . Cell lysates were prepared and then immunoblotted with anti ‐ ATP8A1 antibody . α ‐ tubulin was used as a loading control . C , D COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h . siRNA ‐ resistant GFP ‐ ATP8A1 ( WT or E191Q ) and myc ‐ CDC50A were transfected 48 h after siRNA transfection . Cells were pulsed with Alexa 594 ‐ Tfn and chased in medium with unlabeled Tfn . During the chase , cells were imaged every 6 min for 54 min . The fluorescence intensity of Alexa 594 ‐ Tfn is shown as percentage of that at 0 min ( mean ± SEM , n = 4 - 6 ; * * * P 0 . 001 ; Student ' s t ‐ test ) . See Supplementary Fig S2 for the representative images . E , F COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h . Cells were pulsed with Alexa 594 ‐ CTxB for 5 min and chased for 15 or 60 min . Cells were then fixed and stained for GM130 . In ( E ) , representative images are shown . In ( F ) , the Pearson ' s coefficient between CTxB and GM130 after 60 ‐ min chase is shown . Data represent mean ± SD ( n > 26 cells from three independent experiments ) . * * * P 0 . 001 ; two ‐ tailed Student ' s t ‐ test . Scale bars , 10 μm .

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Figure 1A COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for myc‐tag and TfnR, TGN46, GM130, VPS26, CD63, or LAMP2. mCherry‐Rab11 was expressed in a cell in the top row. Nuclei were stained with DAPI (in blue). Pearson's coefficient between GFP‐ATP8A1 and each organelle marker is shown in the merged image (mean ± SD, n > 20 cells from two experiments). Scale bars, 10 μm.B COS‐1 cells were treated with ATP8A1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with anti‐ATP8A1 antibody. α‐tubulin was used as a loading control.C, D COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Cells were pulsed with Alexa 594‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 6 min for 54 min. The fluorescence intensity of Alexa 594‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n = 4-6; ***P 0.001; Student's t‐test). See Supplementary Fig S2 for the representative images.E, F COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 15 or 60 min. Cells were then fixed and stained for GM130. In (E), representative images are shown. In (F), the Pearson's coefficient between CTxB and GM130 after 60‐min chase is shown. Data represent mean ± SD (n > 26 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.


Figure 2A COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for TfnR . Each image represents the maximum intensity z ‐ projection of four confocal slices . Intensified images are shown in the bottom row . Red dashed lines and arrowheads indicate nuclei and TfnR puncta , respectively . Scale bars , 10 μm . B COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for the indicated proteins . Scale bars , 10 μm . C COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h . TfnR ‐ GFP was transfected 48 h after siRNA transfection . Cells were then imaged with an epifluorescent microscope . Selected stills from Supplementary Movie S1 are shown . The asterisks indicate the tips of the tubules of interest . Scale bars , 5 μm . D The effect of increasing levels of 1 , 2 ‐ dioleoyl ‐ PS on the ATPase activity of purified ATP8A1 ( WT and E191Q ) ‐ CDC50A complex . The Km and Vmax for the WT complex were 59 . 1 ± 3 . 5 μM and 38 . 7 ± 0 . 8 μmol ATP / min / mg protein , respectively . E COS ‐ 1 cells were treated with ATP8A1 siRNA1 for 72 h . siRNA ‐ resistant ATP8A1 ( WT or E191Q ) , and myc ‐ CDC50A were transfected 48 h after siRNA transfection . Cells were then fixed and stained for TfnR . The graph shows percentages of cells showing aberrant TfnR ‐ positive tubules as mean ± SD of three independent experiments . At least 50 cells were counted per experiment .

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Figure 2A COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for TfnR. Each image represents the maximum intensity z‐projection of four confocal slices. Intensified images are shown in the bottom row. Red dashed lines and arrowheads indicate nuclei and TfnR puncta, respectively. Scale bars, 10 μm.B COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h and stained for the indicated proteins. Scale bars, 10 μm.C COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. TfnR‐GFP was transfected 48 h after siRNA transfection. Cells were then imaged with an epifluorescent microscope. Selected stills from Supplementary Movie S1 are shown. The asterisks indicate the tips of the tubules of interest. Scale bars, 5 μm.D The effect of increasing levels of 1,2‐dioleoyl‐PS on the ATPase activity of purified ATP8A1 (WT and E191Q)‐CDC50A complex. The Km and Vmax for the WT complex were 59.1 ± 3.5 μM and 38.7 ± 0.8 μmol ATP/min/mg protein, respectively.E COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. siRNA‐resistant ATP8A1 (WT or E191Q), and myc‐CDC50A were transfected 48 h after siRNA transfection. Cells were then fixed and stained for TfnR. The graph shows percentages of cells showing aberrant TfnR‐positive tubules as mean ± SD of three independent experiments. At least 50 cells were counted per experiment.


Figure 3A COS ‐ 1 cells were treated with EHD1 siRNAs for 72 h . Cell lysates were prepared and then immunoblotted with EHD1 antibody . α ‐ tubulin was used as a loading control . B , C Cells were treated with EHD1 siRNA1 for 72 h . Cells were pulsed with Alexa 488 ‐ Tfn and chased in medium with unlabeled Tfn . During the chase , cells were imaged every 3 min for 57 min . In ( B ) , representative images of Alexa 488 ‐ Tfn in control or EHD1 ‐ depleted cells are shown . In ( C ) , the fluorescence intensity of Alexa 488 ‐ Tfn is shown as percentage of that at 0 min ( mean ± SEM , n > 3 cells from three independent experiments ) . * * * P 0 . 001 ; two ‐ tailed Student ' s t ‐ test . Scale bars , 10 μm . B , C Cells were treated with EHD1 siRNA1 for 72 h . Cells were pulsed with Alexa 488 ‐ Tfn and chased in medium with unlabeled Tfn . During the chase , cells were imaged every 3 min for 57 min . In ( B ) , representative images of Alexa 488 ‐ Tfn in control or EHD1 ‐ depleted cells are shown . In ( C ) , the fluorescence intensity of Alexa 488 ‐ Tfn is shown as percentage of that at 0 min ( mean ± SEM , n > 3 cells from three independent experiments ) . * * * P 0 . 001 ; two ‐ tailed Student ' s t ‐ test . Scale bars , 10 μm . D COS ‐ 1 cells were treated with EHD1 siRNA1 for 72 h . Cells were pulsed with Alexa 594 ‐ CTxB for 5 min and chased for 60 min . Cells were then fixed and stained for GM130 . The Pearson ' s coefficient between CTxB and GM130 is shown ( mean ± SD , n > 30 cells from two independent experiments ) . * * * P 0 . 001 ; two ‐ tailed Student ' s t ‐ test . See Supplementary Fig S7A for the representative images . E COS ‐ 1 cells were treated with EHD1 siRNA1 for 72 h and stained for TfnR . The image represents the maximum intensity z ‐ projection of four confocal slices . Scale bar , 10 μm . F COS ‐ 1 cells co ‐ transfected with GFP ‐ ATP8A1 and myc ‐ CDC50A were stained for EHD1 . Scale bar , 10 μm . G Cells were treated with ATP8A1 siRNA1 for 72 h and stained for EHD1 . For the rescue experiments , siRNA ‐ resistant GFP ‐ ATP8A1 ( WT or E191Q ) and myc ‐ CDC50A were transfected 48 h after siRNA transfection . Asterisks indicate cells with GFP ‐ ATP8A1 expression . The graph represents percentage of cells with perinuclear localization of EHD1 as mean ± SD of three independent experiments ( at least 141 cells were counted for each condition ) . Scale bars , 10 μm .

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Figure 3A COS‐1 cells were treated with EHD1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with EHD1 antibody. α‐tubulin was used as a loading control.B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.D COS‐1 cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 60 min. Cells were then fixed and stained for GM130. The Pearson's coefficient between CTxB and GM130 is shown (mean ± SD, n > 30 cells from two independent experiments). ***P 0.001; two‐tailed Student's t‐test. See Supplementary Fig S7A for the representative images.E COS‐1 cells were treated with EHD1 siRNA1 for 72 h and stained for TfnR. The image represents the maximum intensity z‐projection of four confocal slices. Scale bar, 10 μm.F COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for EHD1. Scale bar, 10 μm.G Cells were treated with ATP8A1 siRNA1 for 72 h and stained for EHD1. For the rescue experiments, siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Asterisks indicate cells with GFP‐ATP8A1 expression. The graph represents percentage of cells with perinuclear localization of EHD1 as mean ± SD of three independent experiments (at least 141 cells were counted for each condition). Scale bars, 10 μm.


Figure 4A The effect of various lipids on the ATPase activity of the purified ATP8A1 ‐ CDC50A complex . The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids : 1 , 2 ‐ dioleoyl ‐ phosphatidycholine ( PC ) , 1 , 2 ‐ dioleoyl ‐ PS , 1 , 2 ‐ dioleoyl ‐ PE , 1 , 2 ‐ dioleoyl ‐ phosphatidylglycerol ( PG ) , 1 , 2 ‐ dioleoyl ‐ phosphatidylinositol ( PI ) , 1 , 2 ‐ dioleoyl ‐ phosphatidic acid ( PA ) , sphingomyelin ( SM ) , cholesterol ( Chol ) , or brain polar lipid ( BPL ) and 90 % PC . The activity of the ATP8A1 ( E191Q ) ‐ CDC50A in the presence of 1 , 2 ‐ dioleoyl ‐ PS was also determined . B NBD ‐ labeled PS or PE transport ( flippase ) activity of ATP8A1 ( WT and E191Q ) ‐ CDC50A complex . The percentage of NBD ‐ lipid flipped was shown . D , E PSA ‐ 3 and R ‐ 41 cells were transiently transfected with GFP ‐ EHD1 and then cultured under the indicated conditions for 72 h . Cells were then fixed , and the nuclei were strained with DAPI . Arrowheads indicate the perinuclear RE localization of GFP ‐ EHD1 . Scale bars , 10 μm .

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Figure 4A The effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex. The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids: 1,2‐dioleoyl‐phosphatidycholine (PC), 1,2‐dioleoyl‐PS, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol (PG), 1,2‐dioleoyl‐phosphatidylinositol (PI), 1,2‐dioleoyl‐phosphatidic acid (PA), sphingomyelin (SM), cholesterol (Chol), or brain polar lipid (BPL) and 90% PC. The activity of the ATP8A1 (E191Q)‐CDC50A in the presence of 1,2‐dioleoyl‐PS was also determined.B NBD‐labeled PS or PE transport (flippase) activity of ATP8A1 (WT and E191Q)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.D, E PSA‐3 and R‐41 cells were transiently transfected with GFP‐EHD1 and then cultured under the indicated conditions for 72 h. Cells were then fixed, and the nuclei were strained with DAPI. Arrowheads indicate the perinuclear RE localization of GFP‐EHD1. Scale bars, 10 μm.


Figure 5A His ‐ tagged EHD1 was mixed with PC ‐ based liposomes containing the indicated % ( mol / mol ) of PS or PE . After 15 min , the mixture was spun at 100 , 000 g for 30 min , and the resultant supernatant ( S ) and pellet ( P ) were subjected to SDS - PAGE . The proteins and lipids were then stained with Coomassie blue . B , C The intensities of individual bands in ( A ) were quantified with ImageJ , and the percentage of bound proteins was calculated . In ( B ) , the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one ‐ way analysis of variance ( ANOVA ) followed by Tukey - Kramer post hoc test . * P 0 . 01 ; NS , not significant . In ( C ) , each point represents the mean of two independent experiments with an error bar showing the range of values obtained . The sigmoidal fitting was applied . D , E His ‐ tagged EHD1 was mixed with PC ‐ based liposomes ( 20 , 40 , or 60 % PS ) of decreasing size produced by extrusion through filter pores of the size indicated . After 15 min , the mixture was spun at 100 , 000 g for 30 min , and the resultant supernatant ( S ) and pellet ( P ) were subjected to SDS - PAGE . The proteins and lipids were then stained with Coomassie blue . In ( E ) , the intensities of individual bands were quantified ( mean ± SEM of three independent experiments ) . See Supplementary Fig S8 for the size distributions of the prepared liposomes .

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Figure 5A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue.B, C The intensities of individual bands in (A) were quantified with ImageJ, and the percentage of bound proteins was calculated. In (B), the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one‐way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. *P 0.01; NS, not significant. In (C), each point represents the mean of two independent experiments with an error bar showing the range of values obtained. The sigmoidal fitting was applied.D, E His‐tagged EHD1 was mixed with PC‐based liposomes (20, 40, or 60% PS) of decreasing size produced by extrusion through filter pores of the size indicated. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue. In (E), the intensities of individual bands were quantified (mean ± SEM of three independent experiments). See Supplementary Fig S8 for the size distributions of the prepared liposomes.


Figure 6A His ‐ tagged 2xPH was mixed with liposomes that were composed of 75 % ( mol / mol ) PE , 20 % ( mol / mol ) PC , and 5 % ( mol / mol ) of PS , PA , PI , PIPs , or sulfatide . In the case of PE / PC , liposomes of 80 % ( mol / mol ) PE and 20 % ( mol / mol ) PC were used . After 15 min , the mixture was spun at 100 , 000 g for 30 min , and the resultant supernatant ( S ) and pellet ( P ) were subjected to SDS - PAGE . The gels were stained with Coomassie blue . B COS ‐ 1 cells were incubated in medium containing Alexa 488 ‐ Tfn for 40 min , washed , fixed , and stained with recombinant 2xPH . Magnified image is shown in the right panel . Scale bar , 10 μm . C COS ‐ 1 cells were transiently transfected with GFP ‐ lact ‐ C2 and stained for Rab11 . Scale bar , 10 μm . D , E COS ‐ 1 cells were transiently transfected with GFP ‐ lact ‐ C2 and stained with recombinant 2xPH and anti ‐ GM130 antibody . The asterisk indicates a cell expressing GFP ‐ lact ‐ C2 , and the arrowhead indicates a non ‐ expressing cell . The graph in ( E ) shows fluorescence intensity per pixel of recombinant 2xPH stain in RE area , which was delimited by GM130 . Data are normalized to the 2xPH intensity in GFP ‐ lact ‐ C2 ( − ) cells ( mean ± SD , n = 13 cells for each condition ) . Scale bar , 10 μm . F - H COS ‐ 1 cells stably expressing GFP ‐ lact ‐ C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti ‐ GM130 antibody . The intensities of 2xPH staining and GFP ‐ lact ‐ C2 in RE area were measured . The intensity of 2xPH ( G ) and the ratio of 2xPH to GFP ‐ lact ‐ C2 intensities ( H ) are shown . In ( H ) , data are normalized to that of control cells . Data represent mean ± SD and were analyzed with two ‐ tailed Student ' s t ‐ test ( n > 26 cells from three independent experiments ) . * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant . Scale bars , 10 μm .

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Figure 6A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide. In the case of PE/PC, liposomes of 80% (mol/mol) PE and 20% (mol/mol) PC were used. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The gels were stained with Coomassie blue.B COS‐1 cells were incubated in medium containing Alexa 488‐Tfn for 40 min, washed, fixed, and stained with recombinant 2xPH. Magnified image is shown in the right panel. Scale bar, 10 μm.C COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained for Rab11. Scale bar, 10 μm.D, E COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained with recombinant 2xPH and anti‐GM130 antibody. The asterisk indicates a cell expressing GFP‐lact‐C2, and the arrowhead indicates a non‐expressing cell. The graph in (E) shows fluorescence intensity per pixel of recombinant 2xPH stain in RE area, which was delimited by GM130. Data are normalized to the 2xPH intensity in GFP‐lact‐C2 (−) cells (mean ± SD, n = 13 cells for each condition). Scale bar, 10 μm.F-H COS‐1 cells stably expressing GFP‐lact‐C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti‐GM130 antibody. The intensities of 2xPH staining and GFP‐lact‐C2 in RE area were measured. The intensity of 2xPH (G) and the ratio of 2xPH to GFP‐lact‐C2 intensities (H) are shown. In (H), data are normalized to that of control cells. Data represent mean ± SD and were analyzed with two‐tailed Student's t‐test (n > 26 cells from three independent experiments). **P 0.01; ***P 0.001; NS, not significant. Scale bars, 10 μm.


Figure 7A COS ‐ 1 cells co ‐ transfected with GFP ‐ ATP8A2 ( WT ) and myc ‐ CDC50A were stained for myc ‐ tag and TfnR . Scale bar , 10 μm . B The effect of increasing levels of PS on the ATPase activity of ATP8A2 ( WT and I376M ) ‐ CDC50A complex . C NBD ‐ labeled PS transport ( flippase ) activity of ATP8A2 ( WT and I376M ) ‐ CDC50A complex . The percentage of NBD ‐ lipid flipped was shown . D Cells were treated with ATP8A1 siRNA1 for 72 h . ATP8A2 ‐ 1D4 ( WT or mutants ) and myc ‐ CDC50A were transfected 48 h after siRNA transfection . The cells were then stained for EHD1 and 1D4 ‐ tag . Scale bars , 10 μm . E Quantification of percentages of cells with perinuclear EHD1 localization . Data represent mean ± SD of three or four independent experiments . At least 20 cells were counted per experiment . F Cells were treated with ATP8A1 siRNA for 72 h . GFP ‐ ATP8A2 ( WT or mutant ) and myc ‐ CDC50A were transfected 48 h after siRNA transfection . The cells were then stained for TfnR . Data represent mean ± SD of three or four independent experiments . At least 20 cells were counted per experiment . G , H Surface biotinylation assay with Atp8a2 + / + or Atp8a2 − / − cortical neurons . The ratio of surface to total TfnR was quantified and normalized to Atp8a2 + / + . Data represent mean value ( 2 mice for each genotype ) .

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

Figure 7A COS‐1 cells co‐transfected with GFP‐ATP8A2 (WT) and myc‐CDC50A were stained for myc‐tag and TfnR. Scale bar, 10 μm.B The effect of increasing levels of PS on the ATPase activity of ATP8A2 (WT and I376M)‐CDC50A complex.C NBD‐labeled PS transport (flippase) activity of ATP8A2 (WT and I376M)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.D Cells were treated with ATP8A1 siRNA1 for 72 h. ATP8A2‐1D4 (WT or mutants) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for EHD1 and 1D4‐tag. Scale bars, 10 μm.E Quantification of percentages of cells with perinuclear EHD1 localization. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.F Cells were treated with ATP8A1 siRNA for 72 h. GFP‐ATP8A2 (WT or mutant) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for TfnR. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.G, H Surface biotinylation assay with Atp8a2+/+ or Atp8a2−/− cortical neurons. The ratio of surface to total TfnR was quantified and normalized to Atp8a2+/+. Data represent mean value (2 mice for each genotype).


Figure 1B . Survival curves of Bcs1l homozygotes without ( GRAC ) and with ( GROX ) alternative oxidase ( AOX ) expression ( n = 18 - 21 / group ) . The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics : The survival data was analyzed using Log - rank ( Mantel - Cox ) test ( p < 0 . 0001 ) C . Weight of mice from P50 to P200 ( n > 10 / group ) and at P600 ( n = 4 - 6 / group ) . D . Blood lactate to glucose ratio at P200 ( n = 8 / group ) Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on graphsDual energy X - ray absorptiometry ( DEXA ) analysis ( n = 10 / group ) of ( E ) lean mass , ( F ) fat mas at P98 Bar graphs represent mean ± SD . The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on graphsDual energy X - ray absorptiometry ( DEXA ) analysis ( n = 10 / group ) o ( G ) bone mineral density at P98 Bar graphs represent mean ± SD . The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on graphsIndirect calorimetric measurement ( n = 10 / group ) of ( H ) heat productio at P77 Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on graphsIndirect calorimetric measurement ( n = 10 / group ) o ( I ) respiratory exchange ratio at P77 Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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

Figure 1B. Survival curves of Bcs1l homozygotes without (GRAC) and with (GROX) alternative oxidase (AOX) expression (n=18-21/group). The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics: The survival data was analyzed using Log-rank (Mantel-Cox) test (p<0.0001)C. Weight of mice from P50 to P200 (n>10/group) and at P600 (n=4-6/group).D. Blood lactate to glucose ratio at P200 (n=8/group) Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphsDual energy X-ray absorptiometry (DEXA) analysis (n=10/group) of (E) lean mass, (F) fat mas at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphsDual energy X-ray absorptiometry (DEXA) analysis (n=10/group) o (G) bone mineral density at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphsIndirect calorimetric measurement (n=10/group) of (H) heat productio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphsIndirect calorimetric measurement (n=10/group) o (I) respiratory exchange ratio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Figure 2A . Hematoxylin - eosin - stained cross sections of heart and kidney at P200 . B . Weight of heart and kidney at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " Sirius Red staining ( C ) for fibrosis in liver , heart and kidney at P200 ( D ) Quantification of fibrosis in myocardium , kidney cortex and liver ( n = 5 - 7 ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " E - F . Echocardiography data ( n = 4 - 6 / group ) showing ( E ) ejection fraction ( EF ) and fractional shortening ( FS ) . ( F ) Systolic ( LV Vol ; S ) and diastolic ( LV Vol ; D ) left ventricle volume in mice Bar graphs represent mean ± SD Statistics One - way ANOVA followed by unpaired t - test with Welch " s correction for graph EG . Expression of cardiac hypertrophy and fibrosis - associated genes in the pre - symptomatic ( P150 ) heart ( n = 6 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " H , I . 24 h excretion of ( H ) albumin ( I ) creatinine in urine at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s tes and Mann - Whitney U tests ( for graph I ) J , K . Liver enzymes ( J ) alanine aminotransferase ( ALT ) and ( K ) alkaline phosphatase ( ALP ) in plasma at P200 ( n = 8 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

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Figure 2A. Hematoxylin-eosin-stained cross sections of heart and kidney at P200.B. Weight of heart and kidney at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"Sirius Red staining (C) for fibrosis in liver, heart and kidney at P200(D) Quantification of fibrosis in myocardium, kidney cortex and liver (n=5-7) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"E-F. Echocardiography data (n=4-6/group) showing (E) ejection fraction (EF) and fractional shortening (FS). (F) Systolic (LV Vol;S) and diastolic (LV Vol;D) left ventricle volume in mice Bar graphs represent mean±SD Statistics One-way ANOVA followed by unpaired t-test with Welch"s correction for graph EG. Expression of cardiac hypertrophy and fibrosis-associated genes in the pre-symptomatic (P150) heart (n=6/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"H, I. 24 h excretion of (H) albumin (I) creatinine in urine at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s tes and Mann-Whitney U tests (for graph I)J, K. Liver enzymes (J) alanine aminotransferase (ALT) and (K) alkaline phosphatase (ALP) in plasma at P200 (n=8/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


Figure 3A . GFAP staining for cerebral astrocytes , the barrel field of the primary somatosensory cortex ( S1BF ) highlighted with bracketsB . Mitochondrial ultrastructure in cardiomyocytes , kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy . Scale bar 1 µm . ( C ) Average cross - sectional area of mitochondrio ( n = 3mice / group Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey " s ( D number of cristae per mitochondrio ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph ) : One - way ANOVA followed by Tukey " s ( E ) cristae thickness , and ( F ) average distance between cristae in mitochondria ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey "

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Figure 3A. GFAP staining for cerebral astrocytes, the barrel field of the primary somatosensory cortex (S1BF) highlighted with bracketsB. Mitochondrial ultrastructure in cardiomyocytes, kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy. Scale bar 1 µm.(C) Average cross-sectional area of mitochondrio (n=3mice/group Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s(D number of cristae per mitochondrio (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph ): One-way ANOVA followed by Tukey"s(E) cristae thickness, and (F) average distance between cristae in mitochondria (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s


Figure 4A - C . Principal component analysis of transcriptome data from ( A ) heart , ( B ) kidney and ( C ) liver . D - F . Heat map visualization of pathway enrichment analysis ( Reactome database ) . Full pathway analysis is provided in Appendix Table S1 . Benjamin - Hochberg FDR - corrected p values are color - labelled as indicated for the three comparisons . Gene expression of major transcriptional regulators of energy metabolism ( PPAR - α , mitochondrial biogenesis ( PGC - 1α , TFAM ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum and minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s test . Gene expression of major transcriptional regulators of energy metabolism HIF - 1α ) and stress responses ( ATF4 ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum and minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s test . L . Expression of cIII stress signature genes in heart , kidney , and liver . ( n = 6 / group )

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

Figure 4A-C. Principal component analysis of transcriptome data from (A) heart, (B) kidney and (C) liver. D-F. Heat map visualization of pathway enrichment analysis (Reactome database). Full pathway analysis is provided in Appendix Table S1. Benjamin-Hochberg FDR-corrected p values are color-labelled as indicated for the three comparisons. Gene expression of major transcriptional regulators of energy metabolism (PPAR-α, mitochondrial biogenesis (PGC-1α, TFAM) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.Gene expression of major transcriptional regulators of energy metabolism HIF-1α) and stress responses (ATF4) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.L. Expression of cIII stress signature genes in heart, kidney, and liver. (n=6/group)


Figure 5A . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in presymptomatic ( P150 ) heart tissue of GRAC ( red ) and GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey " sB . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in liver tissue of GRAC ( red ) and GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey " sC - F . Concentrations of the TCA cycle intermediates ( C ) fumarate and ( D ) malate , ( E ) adenylate energy charge and ( F ) NADH / NAD + ratio ( n = 5 / group ) Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey " s ( G ) Proline concentratio in heart tissue at P200 ( # below detection limit Bar graph represent mean ± SmRNA expression of proline synthesis - related genes ( n = 6 / group ) ) ( H ) Aldh18a1 and ( I ) Pycr1 at P150 box plots of mRNA expressions represent quartiles , minimum , and maximum value ( relative fold change " FC & qu Statistics for graph one - way ANOVA followed by Tukey "

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Figure 5A. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in presymptomatic (P150) heart tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"sB. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in liver tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"sC-F. Concentrations of the TCA cycle intermediates (C) fumarate and (D) malate, (E) adenylate energy charge and (F) NADH/NAD+ ratio (n=5/group) Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s(G) Proline concentratio in heart tissue at P200 (# below detection limit Bar graph represent mean±SmRNA expression of proline synthesis-related genes (n=6/group)) (H) Aldh18a1 and (I) Pycr1 at P150 box plots of mRNA expressions represent quartiles, minimum, and maximum value (relative fold change "FC&qu Statistics for graph one-way ANOVA followed by Tukey"s


Figure 6A . Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes ( SCs ) B - C . RISP per CORE1 ratio in ( B ) SC1 and ( C ) free cIII2 . Spectrophotometric assay of ( D ) cI , ( E ) cII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on gSpectrophotometric assay o ( F ) cIII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on gG . CIV activity as measured using an oxygraph Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on gH . n - Propyl gallate - sensitive AOX mediated cI and cII - linked state 3 respiration ( n = 7 / group , Mann - Whitney U test ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on gCI - linked ( I state 3 respiration in isolated heart , kidney and liver mitochondria ( n = 8 / group ) We used malate , pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on gcI - and cII - linke state 3 respiration in isolated heart , kidney and liver mitochondria ( n = 8 / group ) we added the CII substrate , succinate , to obtain CI & CII - linked respiration in presence of saturating ADP . Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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

Figure 6A. Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes (SCs)B-C. RISP per CORE1 ratio in (B) SC1 and (C) free cIII2.Spectrophotometric assay of (D) cI, (E) cII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on gSpectrophotometric assay o (F) cIII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on gG. CIV activity as measured using an oxygraph Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on gH. n-Propyl gallate-sensitive AOX mediated cI and cII-linked state 3 respiration (n=7/group, Mann-Whitney U test) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on gCI-linked (I state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) We used malate, pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on gcI- and cII-linke state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) we added the CII substrate, succinate, to obtain CI&CII-linked respiration in presence of saturating ADP. Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


Figure 7A . Amplex Red - based detection of H2O2 emission , a surrogate for respiratory chain - derived ROS , during cI and cII - linked state 3 respiration ( n = 6 / group ) Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey " B . Gene expression related to ROS scavengingC . Total glutathion in heart and liver tissue ( n = 5 / group Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey " D . Urinary isoprostanes per creatinine as a measure of oxidative stress ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : Mann - Whitney U test ( graph DE and F . Representative images ( D ) of tissue sections immunostained with an antibody against 4 - hydroxynonenal ( 4 - HNE ) , a lipid peroxidation marker . ( E ) Quantification of the 4 - HNE immunostaining .

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Figure 7A. Amplex Red-based detection of H2O2 emission, a surrogate for respiratory chain-derived ROS, during cI and cII-linked state 3 respiration (n=6/group) Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"B. Gene expression related to ROS scavengingC. Total glutathion in heart and liver tissue (n=5/group Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"D. Urinary isoprostanes per creatinine as a measure of oxidative stress (n=4/group) Bar graphs represent mean±SD Statistics: Mann-Whitney U test (graph DE and F. Representative images (D) of tissue sections immunostained with an antibody against 4-hydroxynonenal (4-HNE), a lipid peroxidation marker. (E) Quantification of the 4-HNE immunostaining.


Figure 8A . Heatmap visualization of the expressions of selected nitric oxide ( NO ) - related genes . B - E . Gene expression of Ca2 + channel gene ( B ) ryanodin receptor , RYR2 , ( C ) sarco ( endo ) plasmic reticulum Ca2 + - ATPase ( SERCA2 or ATP2A2 ) , ( D ) phospholamban ( PLN ) , and ( E ) endothelial nitric oxide synthase NOS3 ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum and maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on graphsExpression of neuronal nitric oxide synthase ( NOS1 ) mRNA ( F Bar graph represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on graphsExpression of neuronal nitric oxide synthase ( NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane ( G ) H . Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 ( DDAH1 ) ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum and maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on graphsI . Cardia citrulline to arginine ratio ( n = 5 / group ) Bar graph represent mean ± SDJ . Cardiac protein nitrotyrosine quantified from Western blotsK . Nitrite concentration in heart tissue ( n = 4 / group ) Bar graph represent mean ±

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

Figure 8A. Heatmap visualization of the expressions of selected nitric oxide (NO)-related genes.B-E. Gene expression of Ca2+ channel gene (B) ryanodin receptor, RYR2, (C) sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2 or ATP2A2), (D) phospholamban (PLN), and (E) endothelial nitric oxide synthase NOS3 (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphsExpression of neuronal nitric oxide synthase (NOS1) mRNA (F Bar graph represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphsExpression of neuronal nitric oxide synthase (NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane (G)H. Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphsI. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SDJ. Cardiac protein nitrotyrosine quantified from Western blotsK. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD


Figure 1 ( A ) THP1 cells were infected with the shown strains of HSV - 1 ( MOI 3 ) . Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity . ( B ) THP1 cells were treated with 0 . 1 μg / ml of acyclovir ( ACV ) and infected with the KOS HSV - 1 and F HSV - 1 strains ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( C ) THP1 cells were treated with infectious or UV - inactivated HSV - 1 ( KOS ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( D ) MDMs were infected with ICP0 - deficient or revertant HSV - 1 ( KOS ) , MOI 3 . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( E ) THP1 - derived cells deficient for cGAS or STING were infected with the shown strains of HSV - 1 ( MOI 3 ) or stimulated with poly ( I : C ) ( 2 µg / ml ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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Figure 1(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) MDMs were infected with ICP0-deficient or revertant HSV-1 (KOS), MOI 3. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


Figure 2 ( A ) MDMs were infected for 18 h with HSV - 1 ( KOS ) , MOI 1 . Cytosolic viral proteins were detected by mass spectrometry using iTRAQ - labeling . Identified viral proteins are shown . ( B ) MDMs were infected with the KOS or F strains of HSV - 1 ( MOI 3 ) . Cell lysates were harvested 12 hpi , and levels of ICP27 and β - actin were determined by Western blotting . ( C ) HFFs and MDMs were infected with HSV - 1 ( KOS ) MOI 0 . 1 and 1 . 0 . Culture medium was replaced 3 hpi , and isolated 24 hpi for viral plaque assay . Data are presented as means of triplicates + / - st . dev . ( D ) THP1 cells were infected with the indicated strains of HSV - 1 ( MOI 3 ) for 12 hrs . Cell lysates were isolated and levels of ICP27 and β - actin were determined by Western blotting .

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

Figure 2(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.(B) MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting.(C) HFFs and MDMs were infected with HSV-1 (KOS) MOI 0.1 and 1.0. Culture medium was replaced 3 hpi, and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates +/- st.dev.(D) THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 hrs. Cell lysates were isolated and levels of ICP27 and β-actin were determined by Western blotting.


Figure 3 ( A ) THP1 cells were infected with HSV - 1 KOS or an ICP27 - deficient mutant ( ΔICP27 ) on KOS genetic background ( MOI 3 ) . Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity . ( B ) MDMs were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( C ) THP1 - derived cells deficient for cGAS or STING were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( D ) HEK293T cells were transfected with 12 ng STING plasmid DNA , 5 or 25 ng ICP27 plasmid DNA , and reporter gene constructs as indicated ( 2x104 cells per well ) . Reporter gene activity was measured in lysates isolated 24 h post transfection . ( E ) THP1 cells were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) in the presence or absence of acyclovir ( ACV ) . Supernatants were isolated 18 hpi for measurement of type I IFN

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

Figure 3(A) THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.(B) MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(C) THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2x104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post transfection.(E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity


Figure 4 ( A ) THP1 cells were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection , and levels of ICP27 , RCC1 , and GAPDH were determined by Western blotting . ( B ) THP1 cells were infected with HSV - 1 KOS or ΔICP27 ( MOI 10 ) . The cells were fixed at the indicated time points post infection , and stained with DAPI and an antibody against ICP27 , and visualized by confocal microscopy . Scale bar 10 μm . ( C , D ) THP1 cells were infected with HSV - 1 KOS , ΔICP27 or HSV - 1 ΔNLS . The localization of the major NLS is illustrated in panel C . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( E , F ) THP1 cells were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Cytoplasmic ( E ) and nuclear extracts ( F ) were isolated at the indicated time points post infection . Western blotting was used to determine levels of phospho - and total TBK1 were determined in the cytoplasmic fractions and levels of phospho - IRF3 and RCC1 in the nuclear fractions . ( G - I ) HEK293T cells were ( G ) transfected with STING , TBK1 , IRF3 - 5D , and ICP27 , together with the reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection . ( G - I ) HEK293T cells were ( ( H ) transfected with STING and stimulated for 16 h with cGAMP ( 4 μg / ml ) together with the reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection . ( G - I ) HEK293T cells were ( I ) transfected with TRIF , MAVS , STING , and ICP27 , together with the reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection .

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Figure 4(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.(B) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post infection, and stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar 10 μm.(C, D) THP1 cells were infected with HSV-1 KOS, ΔICP27 or HSV-1 ΔNLS. The localization of the major NLS is illustrated in panel C. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(E, F) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post infection. Western blotting was used to determine levels of phospho- and total TBK1 were determined in the cytoplasmic fractions and levels of phospho-IRF3 and RCC1 in the nuclear fractions.(G-I) HEK293T cells were (G) transfected with STING, TBK1, IRF3-5D, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.(G-I) HEK293T cells were ((H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.(G-I) HEK293T cells were (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.


Figure 5 ( A ) Lysates from THP1 cells infected with HSV - 1 KOS ( MOI 10 ) were subjected to immunoprecipitation using an anti - STING antibody , and the presence of TBK1 , STING , and ICP27 in the precipitates was detected by Western blotting . ( B , C ) THP1 cells were infected with HSV - 1 KOS ( MOI 10 ) for 6 and 8 h . The cells were fixed and stained with DAPI and anti - ICP27 together with anti - pTBK1 ( B ) . The stainings were visualized by confocal microscopy . Arrowheads , colocalizations between ICP27 and pTBK1 . Scale bar 10 μm . ( B , C ) THP1 cells were infected with HSV - 1 KOS ( MOI 10 ) for 6 and 8 h . The cells were fixed and stained with DAPI and anti - ICP27 together with anti - STING ( C ) . The stainings were visualized by confocal microscopy . Arrowheads , colocalizations between ICP27 and STING . Scale bar 10 μm . ( D ) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF , MAVS , and STING . The precipitates were immunoblotted for TBK1 , ICP27 , and STING . ( E ) THP1 control and STING KO cells were infected with HSV - 1 KOS ( MOI 10 ) for 6 and 8 h and total lysates were generated . TBK1 was immunoprecipitated , and subjected to Western blotting using antibodies against ICP27 , phospho - TBK1 ( pTBK1 ) , and TBK1 . ( F ) THP1 control and TBK1 KO cells were infected with HSV - 1 KOS ( MOI 10 ) for 8 h and total lysates were generated . STING was immunoprecipitated , and subjected to Western blotting using antibodies against ICP27 and STING . ( G ) THP1 cells were infected with HSV - 1 KOS ( MOI 10 ) for 8 h in the presence or absence of the TBK1 inhibitor BX795 ( BX , 200 nM ) . Total lysates were generated and STING was immunoprecipitated , and subjected to Western blotting using antibodies against ICP27 and STING . ( H ) THP1 cells were infected with HSV - 1 KOS ( MOI 10 ) for 6 and 8 h and total lysates were generated . Phospho - TBK1 was immunoprecipitated , and subjected to Western blotting using antibodies against ICP27 and total TBK1 . ( I ) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING ( either WT or S366A ) . The precipitates were immunoblotted for TBK1 , ICP27 , and STING .

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

Figure 5(A) Lysates from THP1 cells infected with HSV-1 KOS (MOI 10) were subjected to immunoprecipitation using an anti-STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting.(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-pTBK1 (B). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and pTBK1. Scale bar 10 μm.(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and STING. Scale bar 10 μm.(D) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING.(E) THP1 control and STING KO cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27, phospho-TBK1 (pTBK1), and TBK1.(F) THP1 control and TBK1 KO cells were infected with HSV-1 KOS (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.(G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.(H) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. Phospho-TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and total TBK1.(I) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.


Figure 6 ( C ) THP1 cells were infected with HSV - 1 ( KOS ) , HSV - 2 ( HG52 ) , HSV - 1 ΔICP27 , or HSV - 1 ΔICP27 rescued with HSV - 2 ICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( D ) RAW264 . 7 cells were infected with HSV - 1 ( KOS ) , PRV ( vBecker3 ) and the corresponding mutants lacking ICP27 ( UL54 in the case of PRV ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ( F ) THP1 cells were infected with HSV - 1 ( KOS ) , ΔRGG , or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ICP27 expression in the infected cells was determined by Western blotting . ( G ) THP1 cells were infected with HSV - 1 KOS or HSV - 1 ΔRGG ( MOI 10 ) for 8 h and total lysates were generated . STING was immunoprecipitated , and subjected to Western blotting using antibodies against ICP27 and STING .

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

Figure 6(C) THP1 cells were infected with HSV-1 (KOS), HSV-2 (HG52), HSV-1 ΔICP27, or HSV-1 ΔICP27 rescued with HSV-2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(F) THP1 cells were infected with HSV-1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting.(G) THP1 cells were infected with HSV-1 KOS or HSV-1 ΔRGG (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.


Figure 1A - C . SPF C57BL / 6Jmice were co - colonized with E1 and N1 wild type , ( WT , A , n = 5 mice ) N1 T6SS mutant , ( ΔtssC , B , n = 4 ) or N1 complemented ( ΔtssC pTssC , C , n = 5 ) . FecalCFU was quantified for E1 ( closed squares ) and N1 ( open squares ) weekly . D . Four weeks post - colonization , E1 fecal recovery was compared between the N1 WT , ΔtssC , and ΔtssC pTssC groups .

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Figure 1A-C. SPF C57BL/6Jmice were co-colonized with E1 and N1 wild type, (WT, A, n=5 mice) N1 T6SS mutant, (ΔtssC, B, n=4) or N1 complemented (ΔtssC pTssC, C, n=5). FecalCFU was quantified for E1 (closed squares) and N1 (open squares) weekly.D. Four weeks post-colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.


Figure 2B - E . Co - colonization of N1 WT ( B and D , n = 4 mice ) or N1 Δbte2 ( C , n = 4 ) with E1 WT ( B and C ) or E1 overexpressing Bti2a ( E1 pBti2a , D ) . FecalCFU was monitored over time ( B - D ) and E1 CFU compared to N1 WT - E1 WT group at four - weeks post co - colonization ( E ) .

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Figure 2B-E. Co-colonization of N1 WT (B and D, n=4 mice) or N1 Δbte2 (C, n=4) with E1 WT (B and C) or E1 overexpressing Bti2a (E1 pBti2a, D). FecalCFU was monitored over time (B-D) and E1 CFU compared to N1 WT-E1 WT group at four-weeks post co-colonization (E).


Figure 3A and B . Initial colonization of gnotobiotic ( A ) or SPF ( B ) mice ( n = 4 mice per group ) with N1 followed by secondary challenge with N1 ( closed squares ) or E1 ( open squares ) . FecalCFU was determined for the primary and secondary colonization strains through four weeks post - secondary challenges .

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Figure 3A and B. Initial colonization of gnotobiotic (A) or SPF (B) mice (n=4 mice per group) with N1 followed by secondary challenge with N1 (closed squares) or E1 (open squares). FecalCFU was determined for the primary and secondary colonization strains through four weeks post-secondary challenges.


Figure 4A - F . Primary colonization of SPF mice with N2 WT , T6SS mutant ( ΔtssC ) and complemented ( ΔtssC pTssC ) followed by secondary challenge with N1 WT ( A and B , n = 5 mice ) N2 WT ( C and D , n = 5 ) or E1 WT ( E and F , n = 4 ) was performed . A , C and E . FecalCFU for primary and secondary strains was determined for four weeks post - secondary challenge . B , D and F . Selected time points were tested for statistical difference of secondary challenge between groups . This includes four weeks post - secondary challenge ( B ) and three days post - challenge ( D and F ) .

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

Figure 4A-F. Primary colonization of SPF mice with N2 WT, T6SS mutant (ΔtssC) and complemented (ΔtssC pTssC) followed by secondary challenge with N1 WT (A and B, n=5 mice) N2 WT (C and D, n=5) or E1 WT (E and F, n=4) was performed.A, C and E. FecalCFU for primary and secondary strains was determined for four weeks post-secondary challenge.B, D and F. Selected time points were tested for statistical difference of secondary challenge between groups. This includes four weeks post-secondary challenge (B) and three days post-challenge (D and F).