A - D Dot and box - plot showing the levels of each p - tau biomarker in each of the AT groups . Aβ - positive ( A + ) was defined by a CSF Aβ42 / 40 < 0 . 071 and Tau - positive ( T + ) by an Elecsys CSF Mid - p - tau181 > 24 pg / ml . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) . The horizontal dashed line indicates the median of the p - tau biomarker in the A - T - group . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

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A-D Dot and box-plot showing the levels of each p-tau biomarker in each of the AT groups. Aβ-positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau-positive (T+) by an Elecsys CSF Mid-p-tau181 > 24 pg/ml. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). The horizontal dashed line indicates the median of the p-tau biomarker in the A-T- group. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


The graphs represent the z - scores changes of each CSF biomarker as a function of CSF Aβ42 / 40 ratio ( as proxy of disease progression ) using a robust local weighted regression method . The z - scores were calculated using the mean and the SD of each CSF biomarker in the A - T - group as a reference . The solid lines depict the trajectory of each CSF biomarker . The dashed lines depict the trajectories of the plasma biomarkers . The vertical black dashed line indicates the CSF Aβ42 / 40 cutoff for A + . Note that the CSF p - tau biomarkers reach the 2 z - scores ( depicted with an horizontal dashed line ) with the following sequence : Mid - p - tau231 , N - p - tau181 , N - p - tau217 , Mid - p - tau181 and t - tau . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , Neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


A Representative clinical picture of two patients with CAOP syndrome ( 14 - year - old patient 1 and 5 - year - old patient 2 ) .

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A Representative clinical picture of two patients with CAOP syndrome (14-year-old patient 1 and 5-year-old patient 2).


B Haematoxylin and eosin ( H & E ) staining of skin biopsies from patient 1 and healthy controls . Scale bars : 200 µm .

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B Haematoxylin and eosin (H&E) staining of skin biopsies from patient 1 and healthy controls. Scale bars: 200 µm.


A , B Representative transmission electron microscopy ( TEM ) images showing keratinocyte mitochondria ( red arrow ) in patient 1 ( A ) and in the Mbtps1 - conditional knockout ( cKO ) mouse model ( B ) . Scale bars : 2 µm .

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A, B Representative transmission electron microscopy (TEM) images showing keratinocyte mitochondria (red arrow) in patient 1 (A) and in the Mbtps1-conditional knockout (cKO) mouse model (B). Scale bars: 2 µm.


C , D Quantification of the mitochondrial number and morphology in patient 1 ( C ) and the Mbtps1 - cKO mouse model ( D ) . ( C ) Left panel : n = 13 biological replicates of normal individuals , n = 10 biological replicates of patient 1 . Middle and right panel : n = 121 biological samples of normal individuals and patient 1 . ( D ) Left panel : n = 23 biological replicates of Mbtps1 - loxP mice ; n = 15 biological replicates of Mbtps1 - cKO mice . Middle panel and right panel : n = 113 biological replicates of Mbtps1 - loxP mice and Mbtps1 - cKO .

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C, D Quantification of the mitochondrial number and morphology in patient 1 (C) and the Mbtps1-cKO mouse model (D). (C) Left panel: n = 13 biological replicates of normal individuals, n = 10 biological replicates of patient 1. Middle and right panel: n = 121 biological samples of normal individuals and patient 1. (D) Left panel: n = 23 biological replicates of Mbtps1-loxP mice; n = 15 biological replicates of Mbtps1-cKO mice. Middle panel and right panel: n =113 biological replicates of Mbtps1-loxP mice and Mbtps1-cKO.


E Immunofluorescence experiments showed that mutant S1P ( p . Val355Gly and p . Ter1053Arg ) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild - type S1P , which suggested that these variants disrupt its mitochondrial import . The white arrowheads indicate the colocation ( yellow ) of S1P ( green ) and the mitochondrial tracer ( red ) . Scale bars : 20 μm ( panels 1 - 4 ) ; scale bars : 2 μm ( panel 5 ) .

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E Immunofluorescence experiments showed that mutant S1P (p.Val355Gly and p.Ter1053Arg) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild-type S1P, which suggested that these variants disrupt its mitochondrial import. The white arrowheads indicate the colocation (yellow) of S1P (green) and the mitochondrial tracer (red). Scale bars: 20 μm (panels 1-4); scale bars: 2 μm (panel 5).


F Cellular component separation assay showing that S1P was enriched in mitochondria . Biochemical fractionation of the whole cell lysate ( WCL ) , cytosol ( Cyto , tubulin ) , mitochondria ( Mito , COX IV ) , lysosomes ( Lyso , LAMP1 ) , Golgi apparatus ( Golgi , GM130 ) , endoplasmic reticulum ( ER , ERp72 ) , and nucleus ( Nue , Lamin B ) from HaCaT cells .

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F Cellular component separation assay showing that S1P was enriched in mitochondria. Biochemical fractionation of the whole cell lysate (WCL), cytosol (Cyto, tubulin), mitochondria (Mito, COX IV), lysosomes (Lyso, LAMP1), Golgi apparatus (Golgi, GM130), endoplasmic reticulum (ER, ERp72), and nucleus (Nue, Lamin B) from HaCaT cells.


G A component separation assay revealed a lower expression of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) than wild - type S1P in mitochondria .

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G A component separation assay revealed a lower expression of mutant S1P (p.Val355Gly and p.Ter1053Arg) than wild-type S1P in mitochondria.


H The impaired binding of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) to translocase of the outer membrane ( TOM ) 70 and translocase of the inner membrane ( TIM ) 23 was detected by a coimmunoprecipitation ( Co - IP ) assay .

H The impaired binding of mutant S1P (p.Val355Gly and p.Ter1053Arg) to translocase of the outer membrane (TOM) 70 and translocase of the inner membrane (TIM) 23 was detected by a coimmunoprecipitation (Co-IP) assay.


A LC - MS / MS analysis of S1P - binding proteins . Total protein lysate was subjected to coimmunoprecipitation ( Co - IP ) with normal IgG or S1P antibody . The purified protein complex was separated by SDS - PAGE and then subjected to silver staining . The arrows indicate the bands containing S1P , ETFA and ETFB . The differential bands were then analysed by liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) .

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A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).


C S1P interacts with endogenous ETFA and ETFB in HaCaT cells . Total protein lysate was subjected to Co - IP with normal IgG , ETFA ( left panel ) or ETFB ( right panel ) antibody . The interaction between S1P and ETFA / ETFB was then detected by immunoblotting .

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C S1P interacts with endogenous ETFA and ETFB in HaCaT cells. Total protein lysate was subjected to Co-IP with normal IgG, ETFA (left panel) or ETFB (right panel) antibody. The interaction between S1P and ETFA/ETFB was then detected by immunoblotting.


D The direct interaction between S1P and ETFA - ETFB was validated by a GST pull - down assay . Left panel : in vitro - translated S1P was pulled down by purified GST - ETFA fusion protein . Right panel : in vitro - translated S1P was pulled down by purified GST - ETFB fusion protein .

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D The direct interaction between S1P and ETFA-ETFB was validated by a GST pull-down assay. Left panel: in vitro-translated S1P was pulled down by purified GST-ETFA fusion protein. Right panel: in vitro-translated S1P was pulled down by purified GST-ETFB fusion protein.


A Representative immunohistochemical images of S1P , ETFA and ETFB in normal controls and patient 1 . Scale bars : 100 μm .

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A Representative immunohistochemical images of S1P, ETFA and ETFB in normal controls and patient 1. Scale bars: 100 μm.


B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1 . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates of normal controls ; n = 6 technical replicates of patient 1 ) and immunohistochemistry ( lower panel , n = 6 biological replicates ; n = 3 technical replicates ) of the S1P , ETFA and ETFB levels .

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B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates of normal controls; n = 6 technical replicates of patient 1) and immunohistochemistry (lower panel, n = 6 biological replicates; n = 3 technical replicates) of the S1P, ETFA and ETFB levels.


C Representative immunohistochemistry images of S1P , ETFA and ETFB in both Mbtps1 - conditional knockout ( cKO ) and Mbtps1 - loxP mice . Scale bars : 100 μm .

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C Representative immunohistochemistry images of S1P, ETFA and ETFB in both Mbtps1-conditional knockout (cKO) and Mbtps1-loxP mice. Scale bars: 100 μm.


D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates ) and immunohistochemistry ( lower panel , n = 6 biological replicates ) of the S1P , ETFA and ETFB levels in Mbtps1 - cKO mice and Mbtps1 - loxP mice .

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D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates) and immunohistochemistry (lower panel, n = 6 biological replicates) of the S1P, ETFA and ETFB levels in Mbtps1-cKO mice and Mbtps1-loxP mice.


E Cycloheximide ( CHX ) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells . Left panel : Representative western blotting images of the ETFA and ETFB protein levels during CHX chase . Right panel : Quantification of the immunoblotting results corresponding to the left panel ( n = 3 biological replicates ) .

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E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates).


F Mutant S1P ( p . Val355Gly and p . Ter1053Arg ) only weakly interacted with ETFA and ETFB . We constructed Flag - tagged wild - type and mutant S1P HaCaT cell lines and then performed a Co - IP assay with Flag antibody , and the interaction between S1P and ETFA / ETFB was detected by immunoblotting .

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F Mutant S1P (p.Val355Gly and p.Ter1053Arg) only weakly interacted with ETFA and ETFB. We constructed Flag-tagged wild-type and mutant S1P HaCaT cell lines and then performed a Co-IP assay with Flag antibody, and the interaction between S1P and ETFA/ETFB was detected by immunoblotting.


G Wild - type S1P , but not mutant S1P ( p . Val355Gly and p . Ter1053Arg ) , decreased the association between ETFA and ETFB . We performed a Co - IP assay with an ETFA antibody , and the interaction between ETFA and ETFB was detected by immunoblotting .

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G Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), decreased the association between ETFA and ETFB. We performed a Co-IP assay with an ETFA antibody, and the interaction between ETFA and ETFB was detected by immunoblotting.


H Wild - type S1P , but not mutant S1P ( p . Val355Gly and p . Ter1053Arg ) , enhanced the incorporation of FAD into the ETF complex . The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm , indicating the incorporation of FAD in the ETF complex . In the presence of the wild - type S1P protein , the two peaks were shifted by 0 . 02 OD units , whereas the additional mutant S1P ( p . Val355Gly and p . Ter1053Arg ) weakly shifted the two peaks by 0 . 005 - 0 . 01 OD units .

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H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.


Mitochondrial respiration ( OCR ) in control ( Ctrl ) and MBTPS1 - knockout ( KO ) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser . Right subpanels : Quantiﬁcation of basal respiration , maximal respiration , and OCR - coupled ATP production in mitochondrial respiration ( n = 5 biological replicates ) .

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Mitochondrial respiration (OCR) in control (Ctrl) and MBTPS1-knockout (KO) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser. Right subpanels: Quantiﬁcation of basal respiration, maximal respiration, and OCR-coupled ATP production in mitochondrial respiration (n = 5 biological replicates).


B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation ( OXPHOS ) and glycolysis in Ctrl and MBTPS1 - KO HaCaT cells ( n = 4 biological replicates ) .

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B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


C Nonmitochondrial respiration ( ECAR ) was quantified in real time using a Seahorse extracellular flux analyser . Quantification of non - glycolytic acidification , glycolysis , glycolytic capacity , and glycolytic reserve in Ctrl and MBTPS1 - KO HaCaT cells ( n = 4 biological replicates ) .

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C Nonmitochondrial respiration (ECAR) was quantified in real time using a Seahorse extracellular flux analyser. Quantification of non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells . Control and MBTPS1 - KO HaCaT cells were cultured in 6 - well dishes . A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit ( n = 4 biological replicates ) .

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D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells. Control and MBTPS1-KO HaCaT cells were cultured in 6-well dishes. A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit (n = 4 biological replicates).


E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species ( Mito SOX ) in HaCaT cells . Left panel : Representative immunofluorescence images of Mito SOX in HaCaT cells . Scale bars : 100 µm . Right panel : The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells ( n = 4 biological replicates ) .

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E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species (Mito SOX) in HaCaT cells. Left panel: Representative immunofluorescence images of Mito SOX in HaCaT cells. Scale bars: 100 µm. Right panel: The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells (n = 4 biological replicates).


A The S1P deficiency - induced decrease in ETFA and ETFB was significantly reversed by riboflavin ( Rib ) in a concentration - dependent manner in MBTPS1 - KO HaCaT cells . GAPDH was used as a loading control . MBTPS1 - KO cells were initially cultured in DMEM for 24 h and supplemented with 0 , 2 . 5 , 5 , or 10 µM riboflavin for 3 days . The expression of ETFA and ETFB was then detected by immunoblotting .

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A The S1P deficiency-induced decrease in ETFA and ETFB was significantly reversed by riboflavin (Rib) in a concentration-dependent manner in MBTPS1-KO HaCaT cells. GAPDH was used as a loading control. MBTPS1-KO cells were initially cultured in DMEM for 24 h and supplemented with 0, 2.5, 5, or 10 µM riboflavin for 3 days. The expression of ETFA and ETFB was then detected by immunoblotting.


B The S1P deficiency - induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA / B overexpression in HaCaT cells . Left panel : Mitochondrial respiration ( OCR ) was quantified in real time using a Seahorse extracellular flux analyser .

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B The S1P deficiency-induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells. Left panel: Mitochondrial respiration (OCR) was quantified in real time using a Seahorse extracellular flux analyser.


C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA / B overexpression in HaCaT cells ( n = 4 biological replicates ) .

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C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells (n = 4 biological replicates).


D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation . The representative head image of patient 1 before therapy is shown in Figure 1A ( panel 1 ) .

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D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation. The representative head image of patient 1 before therapy is shown in Figure 1A (panel 1).


( a ) Activation of the gB - specific CD8 + T cell hybridoma ( which expresses β - galactosidase as an indicator of T cell activation ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , then incubated for 12 h at 37 ° C with the hybridoma . A595 , absorbance at 595 nm .

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(a) Activation of the gB-specific CD8+ T cell hybridoma (which expresses β-galactosidase as an indicator of T cell activation) by macrophages infected for various times (horizontal axis) with HSV-1, then incubated for 12 h at 37 °C with the hybridoma. A595, absorbance at 595 nm.


b ) Activation of the hybridoma as described in a , with the addition of dimethyl sulfoxide ( DMSO ; negative control ) , bafilomycin A ( Baf ) , brefeldin A ( BFA ) or MG - 132 at 2 h after macrophage infection .

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b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection.


( c ) Activation of the hybridoma as described in a , with the addition of bafilomycin A at 2 h after macrophage infection .

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(c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection.


( d ) Immunofluorescence microscopy of gB ( blue ) and LAMP - 1 ( red ) in HSV - 1 - infected macrophages ; pink indicates colocalization . Original magnification , × 40 .

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(d) Immunofluorescence microscopy of gB (blue) and LAMP-1 (red) in HSV-1-infected macrophages; pink indicates colocalization. Original magnification, × 40.


( e ) CD8 + T cell - stimulatory capacity ( as described in a ) of uninfected macrophages ( Mock ) , of BMA ( BMA - HSV ) or J774 ( J774 - HSV ) macrophages infected for 8 h with HSV - 1 , and of cocultures of J774 macrophages ( H - 2d ) infected for 1 h with HSV - 1 , then mixed with uninfected BMA ( H - 2b ) macrophages at a ratio of 1 : 1 and cultured together for 8 h ( J774 - HSV + BMA ) .

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(e) CD8+ T cell-stimulatory capacity (as described in a) of uninfected macrophages (Mock), of BMA (BMA-HSV) or J774 (J774-HSV) macrophages infected for 8 h with HSV-1, and of cocultures of J774 macrophages (H-2d) infected for 1 h with HSV-1, then mixed with uninfected BMA (H-2b) macrophages at a ratio of 1:1 and cultured together for 8 h (J774-HSV + BMA).


( a ) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2 - 8 h ( left margin ) with HSV - 1 . Original magnification , × 20 .

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(a) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2-8 h (left margin) with HSV-1. Original magnification, × 20.


( b ) Activation of the gB - specific CD8 + T cell hybridoma ( described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , with ( DMEM + 3 - MA ) or without ( DMEM ) the addition of 3 - methyladenine 2 h after infection , then incubated for 12 h at 37 ° C with the hybridoma .

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(b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma.


( c ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control ( Ctrl ) siRNA or Atg5 - specific siRNA , then infected for 8 h or 12 h with HSV - 1 .

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(c) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control (Ctrl) siRNA or Atg5-specific siRNA, then infected for 8 h or 12 h with HSV-1.


( d ) Immunoblot analysis of Atg - 5 in siRNA - treated macrophages .

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(d) Immunoblot analysis of Atg-5 in siRNA-treated macrophages.


( e ) Activation of the hybridoma ( as described in b ) by macrophages infected with HSV - 1 and incubated at 37 ° C ( Basal ) , incubated for 12 h at 39 ° C before being infected with HSV - 1 ( heat shock ( HS ) ) , or treated with rapamycin during HSV - 1 infection ( Rapa ) , with ( + Baf ) or without the addition of bafilmycin A at 2 h after infection .

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(e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection.


( f ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control siRNA or Atg5 - specific siRNA , then infected for 8 h with HSV - 1 , with ( + ) or without ( − ) the addition of rapamycin at 2 h after infection . Results in b , c , e , f are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 8 h at 37 ° C without further treatment ( b , e ) or infected macrophages treated with control siRNA ( c , f ) and are presented in arbitrary units . Data are representative of three independent experiments ( a , d ) or are from three independent experiments ( mean and s . e . m . of triplicate samples ; b , c , e , f ) .

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(f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f).


( a - c ) Immunofluorescence microscopy of uninfected macrophages incubated at 37 ° C ( control ; a ) , subjected to mild heat shock ( b ) or treated with rapamycin ( c ) , then stained with anti - LC3 .

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(a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3.


d ) Immunofluorescence microscopy of the expression of LC3 , gB and GFP ( VP26 ) by macrophages infected for various times ( left margin ) with HSV - 1 . White indicates colocalization .

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d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization.


( e ) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h ( left margin ) with wild - type HSV - 1 ( WT ) or HSV - 1 lacking ICP34 . 5 ( Δ34 . 5 ) . Blue , staining of nuclei with DAPI ( 4 , 6 - diamidino - 2 - phenylindole ) . Original magnification , × 100 ( a - c ) or × 63 ( d , e ) . Results in a - e are representative of three independent experiments .

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(e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a-c) or × 63 (d,e). Results in a-e are representative of three independent experiments.


( f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with wild - type HSV - 1 strain 17 + ( WT 17 + ) or Δ34 . 5 HSV - 1 . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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(f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


( g , h ) Immunoblot analysis ( IB ; g ) and flow cytometry ( h ) of the expression of HSV - 1 proteins ( g ) and gB ( h ) in macrophages infected for various times ( above lanes ( g ) or plots ( h ) ) with wild - type or Δ34 . 5 HSV - 1 . Ctrl , control ( uninfected BMA macrophages ; h ) . Data are representative of two ( g ) or three ( h ) independent experiments .

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(g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments.


( i ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( below graph ) with wild - type or Δ34 . 5 HSV - 1 , with ( + ) or without ( - ) the addition of bafilomycin A at 2 h after infection . Results in f , i are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 6 h with wild - type virus ( f ) or with infected macrophages incubated without bafilomycin ( i ) and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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(i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Electron microscopy of macrophages 10 h after infection with HSV - 1 . ( a ) Arrows indicate membrane - coiled structures emerging from the nucleus of an infected cell . ( b - d ) Four - layered membrane structures formed by coiling of the nuclear membrane . ( e , f ) Glucose - 6 - phosphatase ( black deposits ) on autophagosome - like structures emerging from the nuclear envelope or free in the cytoplasm , and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome - like compartment . N , nucleus ; VP , viral particles . Scale bars , 1 μm ( a ) , 0 . 25 μm ( b - d , f ) or 0 . 4 μm ( e ) . Images are representative of three independent experiments with at least 100 cell profiles in each .

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Electron microscopy of macrophages 10 h after infection with HSV-1. (a) Arrows indicate membrane-coiled structures emerging from the nucleus of an infected cell. (b-d) Four-layered membrane structures formed by coiling of the nuclear membrane. (e,f) Glucose-6-phosphatase (black deposits) on autophagosome-like structures emerging from the nuclear envelope or free in the cytoplasm, and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome-like compartment. N, nucleus; VP, viral particles. Scale bars, 1 μm (a), 0.25 μm (b-d,f) or 0.4 μm (e). Images are representative of three independent experiments with at least 100 cell profiles in each.


Immunoelectron microscopy of macrophages 10 h after infection with HSV - 1 . ( a - c ) Accumulation of LC3 ( a , b ) and gB ( c ) . ( d ) Fusion of four - layered membrane structures and lysosomes preloaded with bovine serum albumin - gold ( BSA - gold ; black dots ) . Original magnification , × 54 , 800 ( a , b ) , × 69 , 000 ( c ) or × 38 , 000 ( d ) . Images are representative of three ( a - c ) or two ( d ) independent experiments .

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Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.


( a ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages exposed to DMSO ( negative control ) , mild heat shock , IL - 1β or IFN - γ .

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(a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ.


( b ) Dansylcadaverin staining of untreated macrophages ( Basal ) or macrophages exposed to mild heat shock , IFN - γ or IL - 1β and infected for 8 h with wild - type HSV - 1 , normalized to the signal obtained in basal conditions and presented in arbitrary units .

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(b) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units.


( c - f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages incubated at 37 ° C ( c ) or exposed to mild heat shock ( d ) , IL - 1β ( e ) or IFN - γ ( f ) and infected for 8 h with wild - type HSV - 1 with the addition of DMSO ( negative control ) , 3 - methyladenine , bafilomycin , brefeldin A or MG - 132 at 2 h after infection . Results in a , c - f are normalized to results obtained for CD8 + T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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(c-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Confirmation of peptide entry into neurons . Cultures were incubated with Bio - TMyc ( 25 µM , 1 h ) ( panel b ) or left untreated ( panel a ) . Arrowheads highlight peptide permeability , detected by fluorescein Avidin D ( green ) , into neurons labeled with neuronal - specific antibody NeuN ( red ) . Peptide is not detected in some neurons ( arrow ) and non - neuronal cells ( asterisk ) . Confocal microscopy images correspond to single sections and are representative of five independent experiments . Scale bar , 10 μm .

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Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm.


Effect of TMyc on neuronal survival . Primary cultures were incubated with TMyc ( 5 , 15 or 25 μM , 30 min ) and subjected to treatment with NMDA ( 100 µM ) and glycine ( 10 µM ) for 4 h . Specific neuronal viability was established and expressed relative to values in cultures with no treatment . Means ± SEM are represented ( n = 8 ) and statistical analysis was performed by ANOVA test followed by post - hoc Tukey HSD test .

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Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.


Effect of TFL457 , TFL482 and TFL541 on TrkB - FL levels . Cultures preincubated with TrkB - FL peptides or TMyc ( 25 µM , 30 min ) , followed by chronic NMDA treatment ( 2 h ) , were compared to cells without peptide . Levels of full - length ( FL ) TrkB were established with panTrkB , an antibody for the extracellular domain that also detects the truncated forms ( tTrkB ) . Quantitation of peptide interference of TrkB - FL downregulation . Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA . Means ± SEM are represented and analysis was performed by ANOVA test followed by post - hoc Tukey HSD test ( * p = 0 . 02 ; n = 4 ) .

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Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).


Effect of TFL457 , TFL482 and TFL541 on neuronal viability after chronic excitotoxicity . Cultures were preincubated with peptides and treated with NMDA for 0 - 6 h as before . Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA . Mean ± SEM of eleven ( TFL457 ) , three ( TFL482 ) and seven ( TFL541 ) independent experiments is given .

Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given.


Effect of TFL518 on TrkB - FL levels . Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457 .

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Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457.


TFL518 effect on neuronal viability . Cultures were treated and analyzed as above . No significant differences were found for TFL518 while neuroprotection by TFL457 was

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TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed


Effect of TFL457 on neuronal viability after acute excitotoxicity . Cells were induced with NMDA ( 50 μM ) and glycine ( 10 μM ) for 1 h and , after agonists removal , culture proceeded for 20 h with DL - AP5 ( 200 μM ) and TMyc or TFL457 ( 15 μM ) . Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented .

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Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.


Effect of TFL457 on Y515 and Y816 phosphorylation . Cultures preincubated with TMyc or TFL457 ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) and analyzed with panTrkB or phosphospecific antibodies . Quantitation of pY515 and pY816 . Mean ± SEM of normalized pY515 ( n = 7 ) or pY816 ( n = 4 ) levels obtained after NMDA - treatment ( 2 h ) are represented relative to those found in cells with the same peptide but no NMDA . Statistical analysis was performed by unpaired Student t test ( * p = 0 . 046 ; n . s . = non - significant ) .

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Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies. Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).


Analysis by immunoprecipitation of TFL457 effect on pY816 levels . Cultures preincubated and treated as before with NMDA ( 2 h ) , were immunoprecipitated with antibodies specific for phosphorylated tyrosine ( pY ) . pY816 was analyzed by WB in immunoprecipates .

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Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.


TFL457 effects on neuronal viability after inhibition of TrkB - FL signaling . Cultures were preincubated ( 30 min ) with inhibitors specific for PI3K ( Wortmannin , 100 nM ) , MAPK / ERK ( UO126 , 300 nM ) or PLCγ ( U - 73122 , 5 μM ) before incubation with TMyc or TFL457 ( 25 μM , 30 min ) . Viability was established 4 h after NMDA treatment . Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( * * * p = 0 . 0001 , * * p = 0 . 009 , * p = 0 . 02 , n . s . = non - significant , respectively for TMyc vs TFL457 in untreated or Wortmannin , UO126 or U - 73122 - treated cells ; * * p = 0 . 008 for untreated vs U - 73122 - treated cells preincubated with TFL457 ; n = 5 ) .

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TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).


Dose - dependent TFL457 effects . Cultures incubated with TMyc o TFL457 ( 5 , 15 o 25 μM , 30 min ) were treated with NMDA ( 2 h ) and compared to cells without peptide or NMDA .

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Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.


Time - course TFL457 effects . Cells preincubated with peptides ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) .

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Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).


Quantitation of peptide effects . Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA . Means ± SEM are given . Results were analyzed by a two - way ANOVA test followed by post - hoc Bonferroni test , comparing TMyc or TFL457 - treated cells for each time - point ( TrkB - FL , * * * p = 0 . 0009 and * p = 0 . 02 ; CREB , * p = 0 . 0149 and * p = 0 . 0148 ; pCREB , * * * p = 0 . 0007 and * * p = 0 . 0012 ; MEF2D , * * p = 0 . 0023 and * * p = 0 . 0047 . All values are respectively for 2 or 4 h ; n = 6 ) .

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Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).


Effect of TFL457 on MEF2 - promoter activity . Cultures transfected with pMEF2 ( two minimal wild type MEF2 elements ) or pMEF2mut ( mutant ) were preincubated with CPPs ( 25 μM , 30 min ) and treated with NMDA ( 100 µM , 2 h ) . Means ± SEM of luciferase activities obtained in excitotoxicity , relative to values found in cells treated with same peptide and no NMDA , are presented .

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Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented.


Effect on CRE - promoter activity . pCRE contains two minimal CREs . Peptide preincubation was as above with or without KG - 501 ( 10 µM ) . Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG - 501 .

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Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501.


Effect on NMDAR - subunit promoters . Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A ( pGluN1 , * p = 0 . 016 ; pGluN2A , * p = 0 . 029 ; n = 14 ) .

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Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14).


Effects on BDNF promoter III or TrkB promoter . Cells transfected with pIIIBDNF ( n = 9 ) , pTrkB ( n = 7 ) or pCRE ( n = 5 ) , as a control , were processed as above but using 20 µM NMDA for 4 h . Data are presented and analyzed as in panel A ( pCRE , * * p = 0 . 0098 ; pIIIBDNF , * p = 0 . 029 ; pTrkB , * p = 0 . 033 ) .

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Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033).


Effects of TFL457 on mRNA levels of CREB / MEF2 - regulated genes . Total RNA was extracted from cultures preincubated with CPPs ( 25 μM , 30 min ) and treated or not with NMDA ( 100 µM , 4 h ) . Levels of mRNA were normalized to NSE ( genes expressed in neurons , left panel ) or GAPDH ( neuronal and glial expression , right panel ) . Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented . Differences found in excitotoxicity were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( GluN1 , * * * p = 0 . 00096 ; GluN2A , p = 0 . 98 ; TrkB - FL , * * * p = 0 . 00037 ; BDNF , * p = 0 . 045 ; n = 8 ) .

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Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).


TFL457 interference of RIP and calpain - processing in excitotoxicity . Cultures were preincubated with peptides ( 25 µM , 30 min ) or left untreated before NMDA addition ( 2 h ) . Different exposures are shown to facilitate visualization of FL , f42 / 39 or f32 . Quantitation of normalized f32 and f42 / 39 levels . Means ± SEM are shown relative to cultures treated with NMDA and no peptide .

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TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32. Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide.


Effect of TFL457 on neuronal viability after calpain inhibition . After 30 min preincubation with calpeptin ( Calp , 10 µM ) and calpain inhibitor III ( CiIII , 10 µM ) , cultures were treated with CPPs and NMDA ( 4 h ) as before . Means ± SEM relative to those obtained in untreated cultures are given .

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Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given.


Effect of TFL457 on neuronal viability after metalloproteinase inhibition . Cultures preincubated with GM6001 ( 10 µM , 30 min ) were treated with CPPs and NMDA as before . Analysis was performed as above ( without GM6001 , * * * p = 0 . 0007 ; with GM6001 , * * * p = 0 . 0006 ; n = 4 ) .

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Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4).


Effect of NMDA on total and cell - surface TrkB - FL levels . Cultures were incubated with NMDA ( 100 µM ) or BDNF ( 100 ng / ml ) for 1 h . Membrane proteins were labeled and purified and compared to corresponding total lysates . TrkB - FL levels are expressed relative to untreated cells .

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Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells.


Effect of TFL457 on the decrease of TrkB - FL surface levels induced by NMDA . Analysis was performed as before in cells incubated with CPPs ( 25 μM , 30 min ) and NMDA - treated ( 1 h ) . Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA .

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Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA.


Effect of endocytosis inhibition on TrkB - FL downregulation . The infarcted area of the ipsilateral hemisphere ( I ) was compared to the corresponding region of the contralateral area ( C ) . Results from representative mice injected with dynasore or vehicle are shown . Different exposures are presented to facilitate visualization of dynasore effects on TrkB - FL and f32 . Quantitation of TrkB - FL and TrkB - T1 in the infarcted area . Normalized protein levels are expressed relative to those of the corresponding contralateral region .

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Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.


Confirmation of Bio - TFL457 delivery to mice cortex . Biotinylated TFL457 ( Bio - TFL457 , 4 nMole / g , retro - orbitally injected ) was detected in coronal sections stained with NeuN and DAPI . Representative confocal microscopy images of cortical areas correspond to single sections . Scale bar , 10 µm .

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Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.


Representative analysis of TrkB - FL at 3 h of damage . Comparison of the infarcted area ( I ) and the corresponding contralateral region ( C ) is shown . Quantitation of TrkB - FL levels . Means ± SEM of normalized TrkB - FL levels in the infarcted area relative to the corresponding contralateral

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Representative analysis of TrkB-FL at 3 h of damage. Comparison of the infarcted area (I) and the corresponding contralateral region (C) is shown. Quantitation of TrkB-FL levels. Means ± SEM of normalized TrkB-FL levels in the infarcted area relative to the corresponding contralateral region


Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457 .

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Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.


Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume . Means ± SEM are given . Differences were analyzed by Student ' s t - test ( * * * p = 0 . 00008 ; n = 14 ) . Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc .

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Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume. Means ± SEM are given. Differences were analyzed by Student's t-test (***p = 0.00008; n = 14). Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc.


Evaluation of balance and motor coordination . Number of contralateral hind paw slips were measured after 24 h of damage . Data spread is presented by box and whisker plots showing interquartile range , median , minimum and maximum values .

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Evaluation of balance and motor coordination. Number of contralateral hind paw slips were measured after 24 h of damage. Data spread is presented by box and whisker plots showing interquartile range, median, minimum and maximum values.


IFN - β promoter activity ( A ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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IFN-β promoter activity (A) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( B ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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ISRE reporter activity (B) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( C ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

IFN-β promoter activity (C) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( D ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

ISRE reporter activity (D) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( E ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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IFN-β promoter activity (E) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( F ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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ISRE reporter activity (F) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( G ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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IFN-β promoter activity (G) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( H ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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ISRE reporter activity (H) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


I ISRE reporter activity in Myc - SIRT5 ( 0 , 100 ng or 200 ng ) - transfected HEK293T cells with or without poly ( I : C ) transfection for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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I ISRE reporter activity in Myc-SIRT5 (0, 100 ng or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


J ISRE reporter activity in co - transfection of Myc - RIG - I ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200ng ) - transfected HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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J ISRE reporter activity in co-transfection of Myc-RIG-I (200 ng) together with Myc-SIRT5 (0, 100 ng or 200ng)-transfected HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


K ISRE reporter activity in co - transfection of Myc - MDA5 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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K ISRE reporter activity in co-transfection of Myc-MDA5 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


L ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

L ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


M ISRE reporter activity in co - transfection of Myc - TBK1 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

M ISRE reporter activity in co-transfection of Myc-TBK1 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


N ISRE reporter activity in co - transfection of HA - IRF3 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

N ISRE reporter activity in co-transfection of HA-IRF3 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


O ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 200ng ) or Myc - SIRT5 - H158Y ( 200ng ) respectively in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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O ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (200ng) or Myc-SIRT5-H158Y(200ng) respectively in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


A Confocal microscopy image of endogenous SIRT5 co - localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti - SIRT5 and anti - MAVS antibodies . Mito , Mitotracker ; Scale bar = 8 µm .

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A Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, Mitotracker; Scale bar = 8 µm.


B - C Co - immunoprecipitation of Myc - SIRT5 with HA - MAVS and vice versa . HEK293T cells were co - transfected with indicated plasmids for 24 h . Anti - Myc ( B ) or anti - HA antibody conjugated agarose beads ( C ) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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B-C Co-immunoprecipitation of Myc-SIRT5 with HA-MAVS and vice versa. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-Myc (B) or anti-HA antibody conjugated agarose beads (C) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies. Data information: IP, immunoprecipitation; TCL, total cell lysates.


D Endogenous interaction between MAVS and SIRT5 . Anti - MAVS antibody was used for immunoprecipitation , and normal mouse IgG was used as a control . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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D Endogenous interaction between MAVS and SIRT5. Anti-MAVS antibody was used for immunoprecipitation, and normal mouse IgG was used as a control. Data information: IP, immunoprecipitation; TCL, total cell lysates.


E Endogenous interaction between MAVS and SIRT5 in the wildtype ( WT ) ( MAVS + / + ) or SIRT5 - deficient ( MAVS - / - ) H1299 cells . Anti - MAVS antibody was used for immunoprecipitation , and the interaction was detected by immunoblotting with anti - SIRT5 antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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E Endogenous interaction between MAVS and SIRT5 in the wildtype (WT) (MAVS +/+) or SIRT5-deficient (MAVS -/-) H1299 cells. Anti-MAVS antibody was used for immunoprecipitation, and the interaction was detected by immunoblotting with anti-SIRT5 antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


F GST pull - down assay for GST - tagged MAVS and His - tagged SIRT5 . GST - tagged MAVS and His - tagged SIRT5 were expressed in Escherichia coli ( BL21 ) , respectively . The association of GST - MAVS with His - SIRT5 was detected by immunoblotting with anti - SIRT5 antibody . GST and GST - MAVS proteins were stained with Coomassie blue .

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F GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue.


H - I Co - immunoprecipitation analysis of HA - SIRT5 with Flag - MAVS - truncated mutants . HEK293T cells were co - transfected with the indicated plasmids . Anti - Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies . Flag - MAVS fragments ( WT : full length ; ΔC1 , 1 - 173 aa ; ΔC2 , 1 - 513 aa ; ΔN1 , 91 - 540 aa ; ΔN2 , 173 - 540 aa ; ΔCARD , Δ10 - 77 aa ; ΔPR , Δ103 - 173 aa ; ΔTM , Δ514 - 535 aa ) . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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H-I Co-immunoprecipitation analysis of HA-SIRT5 with Flag-MAVS-truncated mutants. HEK293T cells were co-transfected with the indicated plasmids. Anti-Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies. Flag-MAVS fragments (WT: full length; ΔC1, 1-173 aa; ΔC2, 1-513 aa; ΔN1, 91-540 aa; ΔN2, 173-540 aa; ΔCARD, Δ10-77 aa; ΔPR, Δ103-173 aa; ΔTM, Δ514-535 aa). Data information: IP, immunoprecipitation; TCL, total cell lysates.


A - B MAVS could be succinylated in vitro . GST - MAVS ( A ) extracted from E . coli or Flag - tagged MAVS ( B ) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . Protein succinylation was detected with anti - pan - succinyl - lysine antibody ; GST - MAVS or Flag - MAVS was stained by Coomassie blue .

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A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue.


C The succinylated residue in MAVS was identified by mass spectrometry analysis .

C The succinylated residue in MAVS was identified by mass spectrometry analysis.


E Dot - blot assay for the specificity of anti - succ - K7 - MAVS antibody . Equal amounts of succinyl - peptides or the control peptides were immunoblotted with the indicated dilutions of anti - succ - K7 - MAVS antibody .

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E Dot-blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody.


GST - MAVS ( F ) extracted from E . coli cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

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GST-MAVS (F) extracted from E.coli cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.


Flag - MAVS ( G ) purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

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Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.


H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5 - intact H1299 cells ( SIRT5 + / + ) ( 3 . 2 vs . 1 . 0 ) . The cell lysates from SIRT5 - deficient H1299 cells or the SIRT5 - intact H1299 cells were immunoprecipitated with anti - MAVS antibody or mouse IgG control , followed by immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


I Reconstitution of WT SIRT5 in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) caused a significant reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 0 . 2 ) ; but overexpression of SIRT5 - H158Y in SIRT5 - / - H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 1 . 2 ) . The SIRT5 - / - H1299 cells were transfected with Flag - SIRT5 or Flag - SIRT5 - H158Y , followed by immunoprecipitating with anti - MAVS antibody or mouse IgG control , and immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


J - K Knockout of Sirt5 increased Mavs succinylation in mouse livers ( J ) and lungs ( K ) . Proteins extracted from livers ( J ) and lungs ( K ) of Sirt5 KO and the wildtype littermates ( n = 3 per group ) were detected by the indicated antibodies . MAVS succinylation was determined by anti - succ - K7 - MAVS antibody , and anti - pan - succinyl - lysine antibody was used as positive controls .

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J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.


A - C Quantitative real time PCR analysis ( qPCR ) of IFNβ ( A ) , CXCL10 ( B ) and IFIT1 ( C ) mRNA in HEK293T cells transfected with the control plasmid ( Myc empty ) or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) for 24 h , followed by infection with or without SeV ( SeV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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A-C Quantitative real time PCR analysis (qPCR) of IFNβ (A), CXCL10 (B) and IFIT1 (C) mRNA in HEK293T cells transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) for 24 h, followed by infection with or without SeV (SeV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


D - F qPCR analysis of IFNβ ( D ) , CXCL10 ( E ) , and IFIT1 ( F ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without SeV ( SeV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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D-F qPCR analysis of IFNβ (D), CXCL10 (E), and IFIT1 (F) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without SeV (SeV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


G - I qPCR analysis of IFNβ ( G ) , CXCL10 ( H ) and IFIT1 ( I ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without VSV ( VSV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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G-I qPCR analysis of IFNβ (G), CXCL10 (H) and IFIT1 (I) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without VSV (VSV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).