( 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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(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.


( 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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(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.


( 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 .

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(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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(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.


A 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 .

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A 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 .

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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 .

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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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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.


A 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 .

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A 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 .

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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 .

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 .

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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 .

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.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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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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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.


A 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 .

A 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 .

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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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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.


A 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 .

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A 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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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.


A 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 .

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A 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 .

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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 .

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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 .

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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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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.


A COS ‐ 1 cells co ‐ transfected with GFP ‐ ATP8A2 ( WT ) and myc ‐ CDC50A were stained for myc ‐ tag and TfnR . Scale bar , 10 μm .

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A 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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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).


B . 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 )

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B. 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 ) .

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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

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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 graphs


Dual 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

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Dual 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 graphs


Dual 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

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Dual 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 graphs


Indirect 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

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Indirect 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 graphs


Indirect 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

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Indirect 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


A . Hematoxylin - eosin - stained cross sections of heart and kidney at P200 .

A. 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 "

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

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 "

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(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

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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 E


G . 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 "

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G. 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 )

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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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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"


A . GFAP staining for cerebral astrocytes , the barrel field of the primary somatosensory cortex ( S1BF ) highlighted with

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A. GFAP staining for cerebral astrocytes, the barrel field of the primary somatosensory cortex (S1BF) highlighted with brackets


B . Mitochondrial ultrastructure in cardiomyocytes , kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy . Scale bar 1 µm .

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B. 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 "

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(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 "

(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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(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


A - 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 .

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A-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 .

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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 .

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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 )

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L. Expression of cIII stress signature genes in heart, kidney, and liver. (n=6/group)


A . 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 "

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A. 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"s


B . 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 "

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B. 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"s


C - 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 "

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C-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 ±

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(G) Proline concentratio in heart tissue at P200 (# below detection limit Bar graph represent mean±S


mRNA 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 "

mRNA 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


A . Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes ( SCs )

A. 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 .

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

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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 g


Spectrophotometric 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

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Spectrophotometric 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 g


G . 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

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G. 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 g


H . 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

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H. 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 g


CI - 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

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CI-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 g


cI - 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

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cI- 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


A . 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 "

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A. 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 scavenging


C . Total glutathion in heart and liver tissue ( n = 5 / group Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey "

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C. 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

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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 D


E 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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E 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.


A . Heatmap visualization of the expressions of selected nitric oxide ( NO ) - related genes .

A. 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

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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 graphs


Expression 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

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Expression 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 graphs


Expression of neuronal nitric oxide synthase ( NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane ( G )

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Expression 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

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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 graphs


I . Cardia citrulline to arginine ratio ( n = 5 / group ) Bar graph represent mean ±

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I. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SD


J . Cardiac protein nitrotyrosine quantified from Western

J. Cardiac protein nitrotyrosine quantified from Western blots


K . Nitrite concentration in heart tissue ( n = 4 / group ) Bar graph represent mean ±

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K. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD


( 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 .

(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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

(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 .

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(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 .

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(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.


( 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 .

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(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 .

O O O O O O O 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) 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 .

(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 .

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(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

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(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


( 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 .

(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 .

(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 .

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(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 .

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

(E, 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 .

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(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 .

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(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 .

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

(G-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.


( 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

(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 .

(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

(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 .

(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.


A - 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 .

A-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 .

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

D. Four weeks post-colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.


B - 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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).


A 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 .

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

A 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.


A - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F. 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).


A . Mice were co - colonized with E1 and either N1 WT ( n = 4 ) or N1 ΔtssC ( n = 3 ) . Five days post - inoculation , fecal RNA was extracted and tested for BFT expression via qRT - PCR .

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

A. Mice were co-colonized with E1 and either N1 WT (n=4) or N1 ΔtssC (n=3). Five days post-inoculation, fecal RNA was extracted and tested for BFT expression via qRT-PCR.


B . Four weeks after co - colonization with E1 and either N1 WT or N1 ΔtssC ( n = 4 mice per group ) , the sera were collected , tested via ELISA for anti - BFT IgG and end point titer calculated .

B. Four weeks after co-colonization with E1 and either N1 WT or N1 ΔtssC (n= 4 mice per group), the sera were collected, tested via ELISA for anti-BFT IgG and end point titer calculated.


C - F . Mice pre - treated with DSS were inoculated with no organisms ( sham ) , E1 only , or E1 competed with N1 WT or N1 ΔtssC . Five days post - inoculation , the ceca were weighed ( C ) and fixed for histopathological examination after sham ( D ) , E1 only ( E ) and E1 - N1 WT ( F ) colonizations . Scale bars denote 100μm ( main image ) and 200μm ( inset ) .

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

C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( A ) CD4 + T cells were isolated from the spleen by negative selection before incubating with anti - CD3 / CD28 antibodies for the indicated time periods . Levels of expression of EGR1 , EGR2 , EGR3 and EGR4 were measured by qPCR .

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T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (A) CD4+ T cells were isolated from the spleen by negative selection before incubating with anti-CD3/CD28 antibodies for the indicated time periods. Levels of expression of EGR1, EGR2, EGR3 and EGR4 were measured by qPCR.


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( B ) WBC were collected from WT , EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition ( gating strategies in Appendix Fig S1 ) . Subclasses of CD8 + , CD4 + and Treg populations were determined as shown .

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

T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (B) WBC were collected from WT, EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition (gating strategies in Appendix Fig S1). Subclasses of CD8+, CD4+ and Treg populations were determined as shown.


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . A Cells were incubated under unstimulated conditions or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies for 0 to 5 days ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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

WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. A Cells were incubated under unstimulated conditions or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies for 0 to 5 days (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . B Dose dependence of EGR4 - dependent differences in proliferation were also determined in both CD4 + and CD8 + cells aft er treatment with anti - TCRβ ( E , F ) or anti - CD3 / CD28 ( G , H ) at the indicated concentrations before FACS analysis ( Appendix Fig S2 ) ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. B Dose dependence of EGR4-dependent differences in proliferation were also determined in both CD4+ and CD8+ cells aft er treatment with anti-TCRβ (E,F) or anti-CD3/CD28 (G,H) at the indicated concentrations before FACS analysis (Appendix Fig S2) (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . C , D Generation analysis was then assessed in WT , EGR1 - / - and EGR4 - / - CD4 + ( C ) or CD8 + ( D ) T cells after a 4 day incubation , under unstimulated conditions , or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies . Generation analysis was determined using FlowJo as depicted in Appendix figure S3 ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . . EGR4 deletion significantly shifted the number of generations as determined by two - way ANOVA ; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions ( p < 0 . 0001 ) .

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

WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. C,D Generation analysis was then assessed in WT, EGR1-/- and EGR4-/- CD4+ (C) or CD8+ (D) T cells after a 4 day incubation, under unstimulated conditions, or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies. Generation analysis was determined using FlowJo as depicted in Appendix figure S3 (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).. EGR4 deletion significantly shifted the number of generations as determined by two-way ANOVA; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions (p < 0.0001).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( A - F ) Cytokine secretion was measured by ELISA after incubating CD4 + ( A , C , E ) and CD8 + ( B , D , F ) T cells for 48 hrs in resting conditions ( A , B ) or stimulated with either anti - TCRβ ( C , D ) or anti - CD3 / CD28 antibodies ( E , F ) .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (A-F) Cytokine secretion was measured by ELISA after incubating CD4+ (A,C,E) and CD8+ (B,D,F) T cells for 48 hrs in resting conditions (A,B) or stimulated with either anti-TCRβ (C,D) or anti-CD3/CD28 antibodies (E,F).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR4KO mice by cell sorting . ( G ) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4 + and CD8 + T cells .

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

CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR4KO mice by cell sorting. (G) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4+ and CD8+ T cells.


CD4 + and CD8 + T cells were isolated from the spleens of WT , and EGR4KO mice by cell sorting . ( H ) Comparison of IFNγ levels in WT vs . EGR4 - / - T cells .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, and EGR4KO mice by cell sorting. (H) Comparison of IFNγ levels in WT vs. EGR4-/- T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( I ) Representative FACS plots of CD4 + T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL - 17 antibodies .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (I) Representative FACS plots of CD4+ T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL-17 antibodies.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( J ) Comparison of the number of Th1 polarized cells between WT , EGR1 - / - and EGR4 - / - CD4 + T cells .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (J) Comparison of the number of Th1 polarized cells between WT, EGR1-/- and EGR4-/- CD4+ T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( K ) CD4 + T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (K) CD4+ T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA.


( A ) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4 + and CD8 + T cells isolated by negative selection from WT , EGR1KO and EGR4KO mice . Cells were stimulated with anti - CD3 / CD28 antibodies for the indicated time periods before fixation and staining . Nuclear localization of NFATc1 in CD4 + and CD8 + T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis ( mean ± SEM ; data are based upon 13 to 20 cells / data point from one experiment ; experiment was repeated 3 times ) . EGR4 - mediated differences in NFATc1 localization were determined by two - way ANOVA . NFAT localization was dependent upon EGR4 expression in both CD4 + ( p = 0 . 025 ) and CD8 + ( p = 0 . 039 ) T cells . Post - hoc analysis revealed EGR4 - dependent differences at specific timepoints * p < 0 . 05 ; * * * p < 0 . 001 .

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

(A) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4+ and CD8+ T cells isolated by negative selection from WT, EGR1KO and EGR4KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for the indicated time periods before fixation and staining. Nuclear localization of NFATc1 in CD4+ and CD8+ T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis (mean±SEM; data are based upon 13 to 20 cells/data point from one experiment; experiment was repeated 3 times). EGR4-mediated differences in NFATc1 localization were determined by two-way ANOVA. NFAT localization was dependent upon EGR4 expression in both CD4+ (p=0.025) and CD8+ (p=0.039) T cells. Post-hoc analysis revealed EGR4-dependent differences at specific timepoints * p < 0.05; *** p < 0.001.


( B ) WT , EGR1KO and EGR4KO CD4 + T cells were isolated from the spleen by negative selection before plating on poly - lysine ( control ) or CD3 / CD28 and loading with fura2 . Ca2 + levels shown are from representative single cells measured from multiple experiments . ( C ) Scatter plots showing area under the curve ( AUC ) and variance for each cell measured under all conditions .

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(B) WT, EGR1KO and EGR4KO CD4+ T cells were isolated from the spleen by negative selection before plating on poly-lysine (control) or CD3/CD28 and loading with fura2. Ca2+ levels shown are from representative single cells measured from multiple experiments. (C) Scatter plots showing area under the curve (AUC) and variance for each cell measured under all conditions.


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( A ) CD4 + and CD8 + T cells were loaded with Fura - 2AM and plated on poly - D - lysine ( 2 hrs ) before treating with biotinylated anti - CD3ε , followed by streptavidin where indicated . The average response of the majority of CD4 + ( WT 74 . 2 ± 3 . 2 % ; EGR4 - / - 77 . 8 ± 2 . 9 % ) and CD8 + ( WT 79 . 4 ± 3 . 5 % ; EGR4 - / - 78 . 2 ± 2 . 5 % ) cells that responded to streptavidin crosslinking of CD3 ( mean ± SEM ; 40 - 60 cells / coverslip ; 7 to 8 separate experiments ) .

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

CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments).


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( B ) Cells were plated on anti - CD3 / CD28 antibodies for 2 hrs ; BTP2 ( 10 μM ) was added where marked by the arrow .

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CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( C ) CD4 + T cells were incubated with anti - CD3 / CD28 antibodies for 6 hrs before measuring expression of IFNγ , IL - 2 , IL - 17 and IL - 4 by FACS analysis . Data are mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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CD4+ T cells were isolated by negative selection from WT or EGR4-/-mice. (C) CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 6 hrs before measuring expression of IFNγ, IL-2, IL-17 and IL-4 by FACS analysis. Data are mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( A ) WT or EGR4 - / - cells were loaded with Fura - 2AM and plated on anti - CD3 / CD28 antibodies for 2 hrs . Senicapoc ( 4 μM ) or Tram - 34 ( 5 μM ) were added where marked by the arrow .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( B ) CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice and incubated with anti - CD3 / CD28 antibodies for 6 hrs in the presence or absence of either senicapoc , TRAM - 34 or Shk - Dap22 before measuring expression of IFNγ levels by FACS analysis . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates . * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( C ) To measure the effect of KCa3 . 1 inhibition of cell proliferation , WT and EGR4 - / - CD4 + T cells were isolated from the spleen by cell sorting and stained with CFSE . Cells were incubated with anti - TCRβ antibodies in the presence of vehicle , Senicapoc ( 5 µM ) or TRAM - 34 ( 5 µM ) for 4 days and then collected for FACS analysis . ( D ) Generation analysis was completed for all data using FlowJo software . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( A ) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections .

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B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( B - D ) Mice were sacrificed on day 20 and tumors were counted . Data is presented as total / mouse ( B ) , N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . Mice were sacrificed on day 20 and tumors were counted . ( B ) , lung tumors ( C ) and metastatic tumors ( all tumors found outside of the lungs ; D ) . N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . WT lung tumors were 5 . 407 ± 1 . 843 % CD45 + , while EGR4KO mice were 12 . 445 ± 2 . 239 % CD45 + . WT spleens were 83 . 92 ± 3 . 344 % CD45 + , while EGR4KO mice were 95 . 13 ± 1 . 856 % CD45 + . Relative distributions of T and myeloid cells within the CD45 + populations were determined by flow cytometric analysis . ( E , F ) Distributions of T and myeloid cells found within lung tumors ( E ) vs . the spleen ( F ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F).