Retinalmicroglia were depleted in rd10 / CreDTAmice by oral administration of tamoxifen ( in corn oil ) to activate microglia - specific Cre - mediated recombination diphtheria toxin expression ; control CreDTA were administered corn oil without tamoxifen . A - D Depletion of retinalmicroglia in rd10 retina . Representative retinal section from a P28 control demonstrates Iba1 + microglia in retina , including those infiltrating ONL ( A ) , while a tamoxifen - administered littermate ( B ) was substantially depleted of retinalmicroglia . Scale bar , 40 μm . Microglial in entire retina ( C ) in ONL only ( D ) confirmed efficient depletion of infiltrating microglia following tamoxifen administration ( n = 8 control 11 depleted from four litters , two - sided unpaired t - ) . E - J Effect of microglial depletion on retinal degeneration at P28 - 29 . ONL atrophy thinning in control ( E ) was significantly more advanced relative to microglia - depleted ( F ) . Scale bar , 40 μm . of mean ONL thickness ( G ) mean number of layers of ONLnuclei ( H ) at P28 - 29 demonstrate significantly greater ONL preservation in depleted retinas ; degree of ONL preservation correlated with extent of microglia depletion ( I ) . ( J ) mean density of TUNEL + nuclei in ONL was not significantly decreased in depleted vs . control ( n = 8 control 11 depleted from four litters , two - sided unpaired t - ) . K - N Continuation of microglial depletion until P37 - 39 resulted in persistence of morphological rescue ( K - M ) , with a significant reduction in TUNEL + nuclei density ( N ) ( n = 6 control nine depleted from two litters , two - sided unpaired t - ) . O - R Similar rescue effects as in ( K - N ) remained apparent when depletion was sustained until P50 ( O - R ) , a time when rod degeneration in rd10 model is relatively complete ( n = 9 control 10 depleted from three litters , two - sided unpaired t - ) . S , T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased - ( S ) - adapted ( T ) responses in ERG testing in depleted ( green lines ) relative to control ( black lines ) , in both a - b - wave amplitudes across multiple flash intensities ( n = 9 control nine depleted , * P < 0 . 05 in one - way ANOVA with Sidak ' s multiple comparison ) .

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Retinalmicroglia were depleted in rd10/CreDTAmice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.A-D Depletion of retinalmicroglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinalmicroglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).E-J Effect of microglial depletion on retinal degeneration at P28-29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONLnuclei (H) at P28-29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).K-N Continuation of microglial depletion until P37-39 resulted in the persistence of morphological rescue (K-M), with a significant reduction in TUNEL+nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).O-R Similar rescue effects as in (K-N) remained apparent when depletion was sustained until P50 (O-R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak's multiple comparison test).


A , B Effect of phagocytosis inhibition on retinal microglia ex vivo . Retinal explants acutely isolated from P21 - 23 rd10 / CX3CR1GFP / + mice were incubated in Ringer ' s solution ( control ) , or Ringer ' s solution containing either vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD ( 400 μM ) for 1 h . GFP - labeled ( green ) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes . No morphological changes were detected in microglia incubated in cRAD ( insets show morphologies at higher magnification ) . Scale bar , 40 μm . Quantifications of total number of phagosomes per 40 × field ( left ) mean number of phagosomes per microglia ( right ) demonstrate significant reductions in phagocytic activity of cRGD , relative to cRAD , exposure ( n = 8 imaging fields from in each condition ) .

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A, B Effect of phagocytosis inhibition on retinal microglia ex vivo. Retinal explants acutely isolated from P21-23 rd10/CX3CR1GFP/+ mice were incubated in Ringer's solution (control), or Ringer's solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD (400 μM) for 1 h. GFP-labeled (green) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes. No morphological changes were detected in microglia incubated in cRAD (insets show morphologies at higher magnification). Scale bar, 40 μm. Quantifications of total number of phagosomes per 40× field (left) and the mean number of phagosomes per microglia (right) demonstrate significant reductions in phagocytic activity of cRGD, relative to cRAD, exposure (n = 8 imaging fields from in each condition).


C - N Structural effects of in vivo inhibition of microglial phagocytosis . P20 somatamice were injected intravitreally in one eye with phagocytosis inhibitor cRGD , in control eye with inactive analogue , cRAD . At P23 , prominent microglia infiltration in ONL observed in control - injected eyes ( C ) was decreased in cRGD - injected eyes ( D ) . ONLmicroglia in control eyes demonstrated more numerous phagosomes ( inset , arrows ) ( E ) compared with cRGD - injected eyes ( F ) . Pairwise comparisons of control - vs . cRGD - injected eyes demonstrated that phagocytosis inhibition significantly reduced densities of infiltrating microglia ( G ) microglialphagosomes ( H ) , mean phagosome number per microglia ( I ) . ONL atrophy in control eyes ( J ) was more advanced compared to cRGD - injected eyes ( K ) , with significantly greater mean ONL thickness ( L ) , ONLnuclear layers ( M ) in cRGD - injected eyes . Mean density of TUNEL + ONLnuclei was lower , with marginal significance ( N ) ( n = 13 ; measurements in cRGD - injected eyes normalized to control eyes , two - sided paired t - ) . Scale bar , 40 μm .

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C-N Structural effects of in vivo inhibition of microglial phagocytosis. P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD, and in the control contralateral eye with the inactive analogue, cRAD. At P23, prominent microglia infiltration in the ONL observed in control-injected eyes (C) was decreased in the contralateral cRGD-injected eyes (D). ONLmicroglia in control eyes demonstrated more numerous phagosomes (inset, arrows) (E) compared with cRGD-injected eyes (F). Pairwise comparisons of control- vs. cRGD-injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia (G) and microglialphagosomes (H), and mean phagosome number per microglia (I). ONL atrophy in control eyes (J) was more advanced compared to cRGD-injected eyes (K), with significantly greater mean ONL thickness (L), and ONLnuclear layers (M) in cRGD-injected eyes. Mean density of TUNEL+ONLnuclei was lower, with marginal significance (N) (n = 13 animals; measurements in cRGD-injected eyes normalized to contralateral control eyes, two-sided paired t-test). Scale bar, 40 μm.


O - T Functional effects of in vivo inhibition of microglial phagocytosis . Representative - adapted ERG recordings at P26 ( following intravitreal injections at P20 P23 ) in a cRGD - injected eye ( red ) relative to control eye ( black ) ( O ) showing that mean a - ( P ) b - wave ( Q ) amplitudes were significantly greater in cRGD - injected vs . control eyes at higher flash intensities . Representative - adapted responses ( R ) illustrating that while mean - adapted a - wave amplitudes ( S ) were slightly but not significantly greater in cRGD - injected eyes , mean b - wave amplitudes ( T ) were significantly greater at higher flash intensities ( n = 13 , two - sided paired t - , * P < 0 . 05 , with exact values alongside ) .

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O-T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).


Microglia infiltrating rd10 retina upregulate IL - 1β expression during degeneration . Iba1 + microglia ( green ) were immunonegative for IL - 1β ( red ) at P18 but become prominently IL - 1β immunopositive at P22 ( arrows ) . Scale bars , 25 μm .

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Microglia infiltrating the rd10 retina upregulate IL-1β expression during degeneration. Iba1+ microglia (green) were immunonegative for IL-1β (red) at P18 but become prominently IL-1β immunopositive at P22 (arrows). Scale bars, 25 μm.


Depletion of infiltrating microglia in rd10 retina decreases IL - 1β . Cytokine in rd10 / CreDTA mouse retinas were assayed following tamoxifen - induced microglial depletion ( from P21 to P37 - 50 , green bars ; n = 9 ) compared with their untreated littermate controls ( bars , n = 8 ; values normalized to control in same litter ) . IL - 1β were significantly lowered following microglial depletion , but IL - 6 , CCL2 , or TNFα were not significantly changed .

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Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37-50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed.


Inhibition of microglial phagocytosis results in decreased microglial IL - 1β expression . Eyes of rd10 were treated with intravitreal injections of cRGD at P20 , analyzed at P23 by immunohistochemistry for Iba1 ( green ) IL - 1β ( red ) , compared to uninjected eyes with eyes injected with inactive analog , cRAD ( n = 3 eyes in each category ) . Immunopositivity was high in infiltrating microglia ( indicated by arrows ) for untreated cRAD - injected control eyes significantly reduced in microglia in cRGD - injected eyes ( upper panel ) , as demonstrated by of IL - 1β expression ( of immunopositivity as a fraction of ONL ) ( lower left panel ) . ONL thickness correlated negatively with extent of IL - 1β immunopositivity ( lower right panel ) . Scale bar , 25 μm .

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Inhibition of microglial phagocytosis results in decreased microglial IL-1β expression. Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20, and analyzed at P23 by immunohistochemistry for Iba1 (green) and IL-1β (red), and compared to uninjected eyes and with eyes injected with inactive analog, cRAD (n = 3 eyes in each category). Immunopositivity was high in infiltrating microglia (indicated by arrows) for untreated and cRAD-injected control eyes and significantly reduced in microglia in cRGD-injected eyes (upper panel), as demonstrated by the quantification of IL-1β expression (area of immunopositivity as a fraction of the ONL) (lower left panel). ONL thickness correlated negatively with the extent of IL-1β immunopositivity (lower right panel). Scale bar, 25 μm.


IL - 1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL - 1 receptor antagonist , anakinra ( from P20 - 22 to P26 - 27 ) in one eye of each rd10 , while eye was injected with PBS as a control . Upper panels show representative retinal sections from control PBS - injected IL - 1RA - injected eyes from same . ONL atrophy in control eyes was more advanced compared to IL - 1RA - injected eyes , with greater mean ONL thickness lower mean density of TUNEL + ONL nuclei in IL - 1RA - injected eyes ( lower panels ) ( n = 15 ; measurements in IL - 1RA - injected eyes normalized to control eyes , paired t - ) . Scale bar , 25 μm .

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IL-1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL-1 receptor antagonist, anakinra (from P20-22 to P26-27) in one eye of each rd10 animal, while the contralateral eye was injected with PBS as a control. Upper panels show representative retinal sections from control PBS-injected and IL-1RA-injected eyes from the same animal. ONL atrophy in control eyes was more advanced compared to IL-1RA-injected eyes, with greater mean ONL thickness and lower mean density of TUNEL+ ONL nuclei in IL-1RA-injected eyes (lower panels) (n = 15 animals; measurements in IL-1RA-injected eyes normalized to contralateral control eyes, paired t-test). Scale bar, 25 μm.


( D ) were treated with sodium arsenite ( 50 µM ) for 45 min , followed by recovery in drug - free medium . Kinetics of SG dissolution are reported . were taken over a time period of 4 hrs every 10 min . Dashed lines = 95 % confidence intervals . Number of counted : 159 ( siRNA control ) ; 166 ( siRNA Hsp90 α + β ) .

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(D) Cells were treated with sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β).


( E ) HeLa - Kyoto were treated with HS at 43 . 5 ˚ C for 1 hr . were then allowed to recover at 37 ° C for 1 hr in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . were fixed , stained for SG marker TIA - 1 of with SGs was counted . Number of counted : 605 ( recovery control ) ; 510 ( recovery VER ) ; 1334 ( recovery GA ) ; 468 ( recovery 17AAG ) . n = 3 - 4 independent experiments , ± . p < 0 . 01 ( One - way ANOVA ) . ( F ) G3BP2 - GFP HeLa - Kyoto were treated with MG132 ( 20 µM ) for 3 hrs , followed by recovery for 2 hrs in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . were fixed of with SGs was counted . Number of counted : 439 ( recovery control ) ; 649 ( recovery VER ) ; 637 ( recovery GA ) ; 649 ( recovery 17AAG ) . n = 3 independent experiments , ± . p < 0 . 0001 ( One - way ANOVA ) .

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(E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted. Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA).


( C ) HeLa were subjected to proximity ligation ( PLA ) using antibodies specific for endogenous Hsp90 DYRK3 . PLA foci nuclei were segmented , PLA foci / were automatically quantified . mean of PLA / foci in incubated with no antibodies ( - ) , only Hsp90 antibody , only DYRK3 antibody both Hsp90 DYRK3 antibodies is shown . incubated with Hsp90 DYRK3 antibodies left untreated were used as control . n = 3 independent experiments , ± ; 368 - 504 counted / sample , p < 0 . 002 between negative controls incubated with Hsp90 DYRK3 antibodies ; p = 10 - 6 between incubated with Hsp90 DYRK3 antibodies left untreated ( control ) or treated for 2 hrs with GA ( 5 µM ) ( One - way ANOVA ) . Scale bar is 10 µm .

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(C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm.


( F ) HeLa were lipofected with a cDNA encoding for GFP or GFP - DYRK3 . 6 hrs post - transfection , were incubated in drug - free medium ( - ) or in presence of GA , 17AAG or VER ( concentrations are shown ) . Total were extracted 16 hrs later . GFP TUBA4A were analyzed by immunoblotting . Representative immunoblotting of 3 independent experiments .

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(F) HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments.


( I ) HeLa were lipofected with a cDNA encoding for GFP - DYRK3 . 24 hrs post - transfection , were treated for 45 min with arsenite ( 50 µM ) alone ( control ) or with GA ( 5 µM ) or 17AAG ( 5 µM ) . were then fixed of expressing GFP - DYRK3 with SGs was counted . Total counted : 462 ( sodium arsenite ) ; 444 ( sodium arsenite GA ) ; 561 ( sodium arsenite 17AAG ) . n = 3 independent experiments , ± ; p < 0 . 001 ( One - way ANOVA ) .

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(I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA).


( A , HeLa were left untreated ( control ) or treated with GA ( 5 µM ) , 17AAG ( 5 µM ) or VER ( 10 µM ) for 8 hrs . were then fixed immunostained for speckle marker SC35 DYRK3 . Automatic segmentation of speckles is on SC35 . An automated imaging was used to quantify of speckles highly enriched for DYRK3 ( using a 1 . 5 as threshold ) . Scale bar is 10 µm .

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(A, HeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3. Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Scale bar is 10 µm.


D ) Representative confocal microscopy of with SC35 - positive bodies ( D ) Scale bar is 10 µm .

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D) Representative confocal microscopy images of cells with SC35-positive bodies (D) Scale bar is 10 µm.


( F ) HeLa were lipofected with a cDNA encoding for GFP - DYRK3 - dN . 24 hrs post - transfection GFP - DYRK3 - dN were either left untreated or exposed to GA ( 5 µM ) for 4 hrs . In untreated ( control ) , GFP - DYRK3 - dN is diffusely distributed in cytosol in nucleus . Upon GA treatment , GFP - DYRK3 - dN forms perinuclear ( PN ) . Representative confocal are shown . Scale bar is 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm.


( I ) mCherry - G3BP1 expressing HeLa were lipofected with cDNAs encoding for GFP - DYRK3 - dN or Sup35 - NM - GFP - DYRK3 - dN . 24 hrs post - transfection were treated for 45 min with sodium arsenite to induce SGs , followed by recovery for 4 hrs in presence of GA ( 5 µM ; rec . GA ) . Representative confocal microscopy are shown . Nucleic acid was stained with DAPI . Scale bar is 10 µm .

O O O O O B-MEASURED_VAR 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

(I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce SGs, followed by recovery for 4 hrs in presence of GA (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.


( C ) Quantitation of recovery after bleach of GFP - DYRK3 in G3BP1 - mCherry HeLa - Kyoto 24 h after transfection . were treated with sodium arsenite ( 50 µM ) for 45 min to induce SGs , which were visualized with G3BP1 - mCherry ; then , sodium arsenite was removed were incubated in drug - free medium or in presence of GA ( 5 µM ) or of GSK ( 5 µM ) . FRAP was performed during stress recovery period . A representative image of treated with arsenite followed by recovery in drug - free medium is shown . Arrowheads dotted circle indicate ROI . Scale bar is 5 µm . mean of 13 FRAP curves ( recovery control ) , 12 FRAP curves ( recovery GA ) 14 FRAP curves ( recovery GSK ) is shown in red ; is shown in gray .

O O O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(C) Quantitation of the fluorescence intensity recovery after bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of GA (5 µM) or of GSK (5 µM). FRAP was performed during the stress recovery period. A representative image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP curves (recovery GSK) is shown in red; the sem is shown in gray.


( B ) Confocal microscopy of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs showing colocalization of FUS with TIAR - positive stress granules . Scale bar is 10 µm . ( C ) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs , followed by recovery in drug - free medium ( + rec . control ) or in presence of 17AAG ( 30 μM ; + rec . 17AAG ) or of GSK ( 5 μM ; + rec . GSK ) for 6 hrs . Quantitation of number of SGs per in P525L FUS eGFP MNs . Number of P525L counted n / condition : 2488 , n = 13 ( arsenite ) ; 1045 , n = 12 ( arsenite + rec . control ) ; 646 , n = 6 ( arsenite + rec . 17AAG ) ; 924 , n = 3 ( arsenite + rec . GSK ) , ± ( One - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(B) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm. (C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec. 17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA).


( I ) Double immunofluorescence labeling using antibodies against DYRK3 FUS in lumbar spinal cord α - MNs of healthy subjects ( control ) fALS carrying p . 521C mutation in FUS gene . Control ( upper panel ) : α - MNs showing uniform ( arrows ) speckled pattern of strong immunoreactivity ( arrowhead ) of DYRK3 , as well as diffuse FUS immunoreactivity . fALS ( p . R521C - FUS ; lower panel ) : surviving α - MNs harboring large FUS ( yellow arrows ) showed markedly reduced DYRK3 immunoreactivity both in cytoplasm as well as in nucleus ( red arrowheads ) . Instead , α - MNs devoid of FUS ( arrows ) showed normal DYRK3 immunoreactivity similar to one of α - MNs from normal controls . Asterix ( * ) represents non - specific lipofuscin granules in one of α - MN in FUS - ALS . Paraffin sections ; scale bars is 50 µm . ( J ) of α - MNs showing low or absent DYRK3 ( upper graphic ) FUS ( lower graphic ) in lumbar spinal cord from five healthy subjects ( control ) five fALS carrying p . 521C mutation in FUS gene . Total number of α - MNs analyzed : 403 ( control ) ; 132 ( fALS ) . n = 5 , ± ( Student ' s t - ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_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 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 B-MEASURED_VAR O O O O B-MEASURED_VAR 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

(I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients carrying the p.521C mutation in the FUS gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus (red arrowheads). Instead, α-MNs devoid of FUS aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm. (J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five fALS patients carrying the p.521C mutation in the FUS gene. Total number of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test).


( B ) Interaction between PLD1 ARL11 / 14 is stronger than with ARF1 / 6 , RhoA . Flag - tagged PLD1 was co - expressed with HA - tagged GTP - bound mutants of small GTPases in HEK293A as indicated . PLD1 was immunoprecipitated with Flag beads , co - precipitated small GTPases were detected with an HA antibody . result is a representative of three biologically independent experiments .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 B-MEASURED_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

(B) Interaction between PLD1 and ARL11/14 is stronger than with ARF1/6, RhoA. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. PLD1 was immunoprecipitated with Flag beads, and the co-precipitated small GTPases were detected with an HA antibody. The result is a representative of three biologically independent experiments.


( C ) ARL11 / 14 co - localize with PLD1 . Flag - tagged PLD1 was co - expressed with HA - tagged GTP - bound mutants of small GTPases in HEK293A as indicated . After 24 h , were fixed with 4 % paraformaldehyde , followed by incubation with anti - Flag anti - HA antibodies . Flag HA - antibodies were detected by anti - mouse anti - rabbit secondary antibodies , respectively . DAPI ( blue ) was used for . Scale bars : 10 μm . result is a representative of two biologically independent experiments .

O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O

(C) ARL11/14 co-localize with PLD1. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. The Flag and HA-antibodies were detected by anti-mouse and anti-rabbit secondary antibodies, respectively. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments.


( D ) Mander ' s overlap coefficient of ( C ) . Mander ' s overlap coefficient calculated using JACoP plugin in ImageJ . Mean ± Standard Deviation ( SD ) ; n = 6 technically independent samples .

O O O O O O O O O O O O O O O O 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) Mander's overlap coefficient of (C). Mander's overlap coefficient calculated using JACoP plugin in ImageJ. Mean± Standard Deviation (SD); n= 6 technically independent samples.


( E ) PLD1 preferentially binds to GTP - form , not GDP - form , of ARL11 . Flag - tagged PLD1 was co - expressed with HA - tagged ARL11Q67L ( GTP - form ) or ARL11T26N ( GDP - form ) mutant in HEK293A . result is a representative of two biologically independent experiments .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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

(E) PLD1 preferentially binds to the GTP-form, not the GDP-form, of ARL11. Flag-tagged PLD1 was co-expressed with HA-tagged ARL11Q67L (GTP-form) or ARL11T26N (GDP-form) mutant in HEK293A cells. The result is a representative of two biologically independent experiments.


( F ) subcellular co - localization of ARL11 with PLD1 is GTP - dependent . HA - tagged GTP - bound ARL11Q67L , wild - type ARL11 or GDP - bound ARL11T26N was co - expressed with Flag - tagged PLD1 . After 24 h , were fixed , followed by incubation with anti - Flag anti - HA antibodies . DAPI ( blue ) was used for . Scale bars : 10 μm . result is a representative of three biologically independent experiments .

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

(F) The subcellular co-localization of ARL11 with PLD1 is GTP-dependent. HA-tagged GTP-bound ARL11Q67L, wild-type ARL11 or GDP-bound ARL11T26N was co-expressed with Flag-tagged PLD1. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments.


( G ) Mander ' s overlap coefficient of ( F ) .

(G) Mander's overlap coefficient of (F).


( B ) loop region of PLD1 is required for interaction between ARL11 PLD1 . Flag - tagged full - or truncations of PLD1 was co - expressed with HA - tagged ARL11Q67L , PLD1 was immunoprecipitated with Flag beads , ARL11 that associated with it was detected with an HA antibody . result is a representative of two biologically independent experiments .

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

(B) The loop region of PLD1 is required for the interaction between ARL11 and PLD1. Flag-tagged full-length or truncations of PLD1 was co-expressed with HA-tagged ARL11Q67L, PLD1 was immunoprecipitated with Flag beads, and ARL11 that associated with it was detected with an HA antibody. The result is a representative of two biologically independent experiments.


( C ) Deletion of loop region disrupts PLD1 ARL11 co - localization . Flag - tagged full - or ∆ Loop truncation of PLD1 was co - expressed with HA - tagged ARL11Q67L . After 24 h , were fixed with 4 % paraformaldehyde , followed by incubation with anti - Flag anti - HA antibodies . DAPI ( blue ) was used for . Scale bars : 10 μm . result is a representative of three biologically independent experiments . ( D ) Mander ' s overlap coefficient of ( J ) . Mander ' s overlap coefficient calculated using JACoP plugin in ImageJ .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O

(C) Deletion of the loop region disrupts the PLD1 and ARL11 co-localization. Flag-tagged full-length or ∆Loop truncation of PLD1 was co-expressed with HA-tagged ARL11Q67L. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments. (D) Mander's overlap coefficient of (J). Mander's overlap coefficient calculated using JACoP plugin in ImageJ.


( B ) ARL11 Knockout of THP - 1 stable pools . ARL11 knockout THP - 1 pools were generated by CRISPR / Cas9 technology with three different guide RNAs . lysates were immunoblotted with indicated antibodies .

O O O B-CONTROLLED_VAR 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

(B) ARL11 Knockout of THP-1 stable cell pools. ARL11 knockout THP-1 cell pools were generated by the CRISPR/Cas9 technology with three different guide RNAs. Cell lysates were immunoblotted with the indicated antibodies.


( D ) THP - 1 pool stably expressing HA - tagged ARL11Q67L or ARL11T26N . Lysates were immunoblotted with indicated antibodies .

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(D) THP-1 cell pool stably expressing HA-tagged ARL11Q67L or ARL11T26N. Lysates were immunoblotted with indicated antibodies.


( E ) ARL11Q67L , but not ARL11T26N , promotes phagocytosis of macrophages . Macrophages were differentiated from indicated THP1 pool , followed by incubated with green zymosan particles for 1 hour . Scale bars : 50 μm . result is a representative of two biologically independent experiments .

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

(E) ARL11Q67L, but not ARL11T26N, promotes phagocytosis of macrophages. Macrophages were differentiated from indicated THP1 cell pool, followed by incubated with green zymosan particles for 1 hour. Scale bars: 50 μm. The result is a representative of two biologically independent experiments.


( F ) result of ( E ) . Data are presented as mean ± SD , n = 8 technically independent samples .

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(F) Quantification result of (E). Data are presented as mean± SD, n = 8 technically independent samples.


( G ) PLD1 knockout efficiency HA - tagged ARL11Q67L expression of THP - 1 stable pools . Lysates were immunoblotted with indicated antibodies .

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(G) PLD1 knockout efficiency and HA-tagged ARL11Q67L expression of THP-1 stable cell pools. Lysates were immunoblotted with indicated antibodies.


( H ) PLD1 knockout blocks ARL11Q67L - stimulated phagocytosis . Macrophages were differentiated from indicated THP1 groups then incubated with green zymosan particles for 1 . 5 hours . Scale bars : 50 μm . result is a representative of two biologically independent experiments . ( I ) PLD1 is required for ARL11 to stimulate phagocytosis . Data are of panel ( H ) .

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

(H) PLD1 knockout blocks the ARL11Q67L-stimulated phagocytosis. Macrophages were differentiated from indicated THP1 cell groups and then incubated with green zymosan particles for 1.5 hours. Scale bars: 50 μm. The result is a representative of two biologically independent experiments. (I) PLD1 is required for ARL11 to stimulate phagocytosis. Data are quantification of panel (H).


( B ) Interaction of PI4KB with ARL5A / 5B is stronger than ARF1 , RAB11A / 11B , RAB14 . Flag - tagged PI4KB was co - expressed with HA - tagged GTP - bound mutant of GTPases in HEK293A as indicated . PI4KB was immunoprecipitated with Flag beads , co - precipitated GTPases were detected by HA Western .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O

(B) Interaction of PI4KB with ARL5A/5B is stronger than ARF1, RAB11A/11B, and RAB14. Flag-tagged PI4KB was co-expressed with HA-tagged GTP-bound mutant of GTPases in HEK293A cells as indicated. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated GTPases were detected by HA Western.


( C ) PI4KB preferentially binds to GTP - form of ARL5B . Flag - tagged PI4KB was co - expressed with HA - tagged ARL5BQ70L ( GTP - form ) or ARL5BT30N ( GDP - form ) mutant in HEK293A . Interaction was measured similar to panel B . result is a representative of two biologically independent experiments .

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(C) PI4KB preferentially binds to the GTP-form of ARL5B. Flag-tagged PI4KB was co-expressed with HA-tagged ARL5BQ70L (GTP-form) or ARL5BT30N (GDP-form) mutant in HEK293A cells. Interaction was measured similar to panel B. The result is a representative of two biologically independent experiments.


( D ) ARL5A colocalization with PI4KB is GTP - dependent . HA - tagged GTP - bound ARL5AQ70L , wild - type ARL5A or GDP - bound ARL5AT30N was co - expressed with Flag - tagged PI4KB . After 24 h , were fixed , followed by incubation with anti - Flag anti - HA antibodies . DAPI ( blue ) was used for . Scale bars : 10 μm . Shown is a representative of two biologically independent experiments . left panel is of overlap coefficient . Mean ± SD ; n = 6 technically independent samples .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_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

(D) The ARL5A colocalization with PI4KB is GTP-dependent. HA-tagged GTP-bound ARL5AQ70L, wild-type ARL5A or GDP-bound ARL5AT30N was co-expressed with Flag-tagged PI4KB. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. Shown is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient. Mean± SD; n= 6 technically independent samples.


( E ) ARL5A / 5B double knockout diminishes colocalization of PI4KB TGN46 . Flag - tagged PI4KB was expressed in HEK293A wild - type or two ARL5A / 5B double knockout clones as indicated . After 24 hours , were fixed , followed by incubation with anti - Flag anti - TGN46 antibodies . DAPI ( blue ) was used for . Scale bars : 5 μm . result is a representative of two biologically independent experiments . left panel is of overlap coefficient .

O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR 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 B-MEASURED_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

(E) ARL5A/5B double knockout diminishes the colocalization of PI4KB and TGN46. Flag-tagged PI4KB was expressed in HEK293A wild-type or two ARL5A/5B double knockout clones as indicated. After 24 hours, cells were fixed, followed by incubation with anti-Flag and anti-TGN46 primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 5 μm. The result is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient.


( G ) N1 region of PI4KB is required for interaction with ARL5B . Flag - tagged full - or truncations of PI4KB were co - expressed with HA - tagged ARL5BQ70L . PI4KB was immunoprecipitated with Flag beads , co - precipitated ARL5B was detected by HA Western . result is a representative of three biologically independent experiments .

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

(G) The N1 region of PI4KB is required for the interaction with ARL5B. Flag-tagged full-length or truncations of PI4KB were co-expressed with HA-tagged ARL5BQ70L. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated ARL5B was detected by HA Western. The result is a representative of three biologically independent experiments.


( H ) N1 region of PI4KB is required for co - localization of ARL5A / B PI4P . P4Mx2 - mEGFP was co - expressed with HA - tagged ARL5AQ70L in HEK293A wild - type or PI4KB knockout ( clone # 2 - 5 ) . HEK293A were co - transfected with indicated constructs . After 24 h , were fixed with 4 % paraformaldehyde , followed by incubation with anti - HA antibodies . DAPI ( blue ) was used for nuclei . Scale bars : 10 μm . result is a representative of two biologically independent experiments . ( I ) Mander ' s overlap coefficient of ( H ) .

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(H) The N1 region of PI4KB is required for the co-localization of ARL5A/B and PI4P. P4Mx2-mEGFP was co-expressed with HA-tagged ARL5AQ70L in HEK293A wild-type or PI4KB knockout cells (clone#2-5). HEK293A cells were co-transfected with indicated constructs. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-HA primary antibodies. DAPI (blue) was used for nuclei staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments. (I) Mander's overlap coefficient of (H).


( B ) ARL5A / 5B recruitment to mitochondria induces PI4P accumulation at mitochondria . Tom20 - CFP - RFB P4Mx2 - mEGFP were co - expressed with RFP - FKBP vector or RFP - FKBP - ARL5A / 5BQ70L constructs in HEK293A . samples were treated with or without 100nM rapamycin for 1 hour . Scale bars : 10 μm . dashed box indicates merged signal of PI4P with ARL5A / 5B or Tom20 . result is a representative of two biologically independent experiments . ( C ) PI4KB knockout blocks PI4P accumulation induced by mitochondria anchored ARL5A / 5B . Tom20 - CFP - RFB P4Mx2 - mEGFP were co - expressed with RFP - FKBP vector or RFP - FKBP - ARL5A / 5BQ70L constructs in HEK293A PI4KB knockout ( clone # 2 - 5 ) . samples were treated with or without 100nM rapamycin for 1 hour before imaging . Scale bars : 10 μm . dashed box indicates merged signal of PI4P with ARL5A / 5B or Tom20 . result is a representative of two biologically independent experiments . (

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(B) ARL5A/5B recruitment to mitochondria induces PI4P accumulation at the mitochondria. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A cells. Cell samples were treated with or without 100nM rapamycin for 1 hour. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (C) PI4KB knockout blocks the PI4P accumulation induced by mitochondria anchored ARL5A/5B. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A PI4KB knockout cells (clone#2-5). Cell samples were treated with or without 100nM rapamycin for 1 hour before imaging. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (


( D ) Mander ' s overlap coefficient of ( B C , result of rapamycin treatment ) . Mean ± SD ; n = 5 technically independent samples .

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(D) Mander's overlap coefficient of (B and C, result of rapamycin treatment). Mean± SD; n= 5 technically independent samples.


( E ) ARL5A / 5B double knockout decreases secretion . Gaussia - luciferase was expressed in HEK293A wild - type or three ARL5A / 5B double knockout clones as indicated . After 24 hours , medium was replaced with fresh medium . After 4 hours , medium was collected , secreted Gaussia - luciferase was detected by Western . were also collected probed with indicated

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(E) ARL5A/5B double knockout decreases protein secretion. Gaussia-luciferase was expressed in HEK293A wild-type or three ARL5A/5B double knockout clones as indicated. After 24 hours, the medium was replaced with the fresh medium. After 4 hours, medium was collected, and the secreted Gaussia-luciferase was detected by Western. Cells were also collected and probed with indicated antibodies


( H ) PI4KB knockout blocks secretion stimulated by ARL5A / 5BQ70L . Experiments were similar to panel ( G ) . Gaussia - luciferase in culture medium was detected by Western lysate was immunoblotted with indicated antibodies .

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(H) PI4KB knockout blocks protein secretion stimulated by ARL5A/5BQ70L. Experiments were similar to panel (G). Gaussia-luciferase in culture medium was detected by Western and the cell lysate was immunoblotted with indicated antibodies.


( A ) Immunofluorescence of HeLa transfected with either a non - target dsRNA ( NT ) or one of three siRNAs targeting LETM1 ( siR1 , siR2 or siR3 ) labeled with anti - TOM20 antibody . In siR1 treated , mitochondria formed a ' honeycomb ' of swollen distinct organelles ; siR3 resulted in giant organelles with a central region distinguished by reduced TOM20 ; siR2 , produced relatively little swelling mitochondrial network was generally well preserved . pronounced swelling induced by siR1 significantly increased circularity ( p = 2 . 32E - 58 ) ( ImageJ ) , siR1 circularity = 0 . 683 ± 0 . 012 compared with 0 . 414 ± 0 . 008 for NT ; where 1 = a perfect circle . Over 150 mitochondria were quantified in each type ; data are means ± . Scale bar represents 12 µm in main 4 µm in offset magnification

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(A) Immunofluorescence analysis of HeLa cells transfected with either a non-target dsRNA (NT) or one of the three siRNAs targeting LETM1 (siR1, siR2 or siR3) and labeled with anti-TOM20 antibody. In siR1 treated cells, the mitochondria formed a 'honeycomb' of swollen distinct organelles; siR3 resulted in giant organelles with a central region distinguished by reduced TOM20; siR2, produced relatively little swelling and the mitochondrial network was generally well preserved. The pronounced swelling induced by siR1 significantly increased circularity (p = 2.32E-58) (ImageJ analysis), siR1 circularity = 0.683 ±0.012 compared with 0.414 ±0.008 for the NT; where 1 = a perfect circle. Over 150 mitochondria were quantified in each cell type; data are means ± SEM. Scale bar represents 12 µm in the main images and 4 µm in offset magnification


( B ) 35S labeling of de novo mitochondrial synthesis of HeLa transfected as in ( A ) . Polypeptide assignments flank gel . Coomassie stained gels are used as loading controls immunoblots indicate efficiency of LETM1 knockdown

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(B) 35S labeling of de novo mitochondrial protein synthesis of HeLa cells transfected as in (A). Polypeptide assignments flank the gel images. Coomassie stained gels are used as loading controls and immunoblots indicate the efficiency of LETM1 knockdown


( D ) Representative immunoblots for OXPHOS NDUFB8 COII of HeLa transfected with NT or siLETM1 ( siR1 , siR2 or siR3 ) . Vinculin GAPDH are shown as loading controls . mean relative abundances for respiratory subunits COII NDUFB8 are shown beneath blots . Data are expressed as mean ± of n = 8 independent experiments

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(D) Representative immunoblots for the OXPHOS proteins NDUFB8 and COII of HeLa cells transfected with NT or siLETM1 (siR1, siR2 or siR3). Vinculin and GAPDH are shown as loading controls. The mean relative abundances for respiratory subunits COII and NDUFB8 are shown beneath the blots. Data are expressed as mean ± SEM of n=8 independent experiments


( E ) Mitochondrial oxygen consumption rate ( OCR ) measured using a Seahorse flux analyzer before ( basal ) after addition ( maximal ) of uncoupler FCCP , in HeLa treated with NT or siLETM1 ( siR1 or siR2 ) . Data are means ± of n = 5 experiments

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(E) Mitochondrial oxygen consumption rate (OCR) measured using a Seahorse flux analyzer before (basal) and after the addition (maximal) of the uncoupler FCCP, in HeLa cells treated with NT or siLETM1 (siR1 or siR2). Data are means ± SEM of n=5 experiments


( A ) LETM1 expression was suppressed in HeLa by transfection with targeted siRNAs ( siR1 , siR2 or siR3 ) . A non - target dsRNA ( NT ) served as control . were fixed immunolabeled with anti - antibody ( green ) . A higher magnification of selected mtDNA foci is shown beside each picture . ( B ) of in ( A ) displaying mtDNA abnormalities . At least 50 per siRNA were counted from 4 ( siR2 ) 5 ( siR1 or siR3 ) independent experiments . Data are expressed as means ± . * * * P < 0 . 001 ( One - way ANOVA )

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(A) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2 or siR3). A non-target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti-DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture. (B) Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as means ± SEM. ***P<0.001 (One-way ANOVA)


( C ) HeLa were labeled with bromouridine ( BrU ) for 60 mins , 144 h after transfection with NT or siR2 stained with anti - BrdU ( green ) anti - GRSF1 ( red ) antibodies . nuclei were stained with DAPI ( blue )

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(C) HeLa cells were labeled with bromouridine (BrU) for 60 mins, 144 h after transfection with NT or siR2 and stained with anti-BrdU (green) and anti-GRSF1 (red) antibodies. Cell nuclei were stained with DAPI (blue)


( D ) HeLa were labeled with anti - ( green ) anti - GRSF1 ( red ) antibodies 144 h after transfection with NT or siR3 . nuclei were stained blue with DAPI

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(D) HeLa cells were labeled with anti-DNA (green) and anti-GRSF1 (red) antibodies 144 h after transfection with NT or siR3. Cell nuclei were stained blue with DAPI


( A ) Steady state of LETM1 in fibroblasts from controls ( C1 - C3 ) , WHS LETM1 + / - ( S1 , S3 , S4 ) S5 sole case of WHS in this study whose deletion does not encompass LETM1 ( boxed in blue ) . GAPDH is shown as a loading control

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(A) Steady state levels of LETM1 protein in fibroblasts from controls (C1-C3), WHS LETM1+/- (S1, S3, S4) and S5 the sole case of WHS in this study whose deletion does not encompass LETM1 (boxed in blue). GAPDH is shown as a loading control


( B ) Immunofluoresence of control ( C1 ) , WHS LETM1 + / - S1 - S4 , fibroblasts with anti - TOM20 antibody . Scale bars are 8 µm in main 3 µm in offset merged magnifications

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(B) Immunofluoresence analysis of control (C1), and WHS LETM1+/- S1-S4, fibroblasts with anti-TOM20 antibody. Scale bars are 8 µm in the main images and 3 µm in offset merged magnifications


( C ) Electron of controls ( C1 C2 ) WHS LETM1 + / + ( S5 ) ; side panels show zooms . Scale bar : 0 . 2 µm . Black arrows indicate areas shown offset at higher magnification ( D ) Electron of WHS LETM1 + / - S1 , S3 S4 . Black arrows indicate elongated branched mitochondria ; side panels show magnifications . Scale bar : 0 . 2 µm

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(C) Electron micrographs of controls (C1 and C2) and WHS LETM1+/+ (S5) cells; side panels show zooms. Scale bar: 0.2 µm. Black arrows indicate the areas shown offset at higher magnification (D) Electron micrographs of WHS LETM1+/- S1, S3 and S4 cells. Black arrows indicate elongated and branched mitochondria; side panels show magnifications. Scale bar: 0.2 µm


( A ) Representative immunoblots of mitochondrial lysates of HEK separated on a 20 - 42 . 5 % iodixanol gradients probed for LETM1 , PDH TFAM

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(A) Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20-42.5% iodixanol gradients probed for LETM1, PDH and TFAM


( B ) Left panel : immunoblot of total PDH inactive phosphorylated ( repressed form ) , PDHS293 , in control ( C1 , C3 ) , WHS LETM1 + / - ( S1 , S3 , S4 ) WHS LETM1 + / + ( S5 ) fibroblasts . Center Right panels : Immunoblot of PDK3 in control ( C1 - C3 ) , WHS LETM1 + / - ( S1 , S3 S4 ) WHS LETM1 + / + ( S5 ) fibroblasts

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(B) Left panel: immunoblot analysis of total PDH protein levels and the inactive phosphorylated (repressed form), PDHS293, in control (C1, C3), WHS LETM1+/- (S1, S3, S4) and WHS LETM1+/+ (S5) fibroblasts. Center and Right panels: Immunoblot analysis of PDK3 in control (C1-C3), WHS LETM1+/- (S1, S3 and S4) and WHS LETM1+/+ (S5) fibroblasts


( C ) Left panel : change in confluence of control ( C1 - C3 ) , WHS LETM1 + / - ( S1 - S4 ) WHS LETM1 + / + ( S5 ) fibroblasts grown in BHB over course of 188 h . Data are expressed as means ± of n = 3 independent experiments . Right panel : of control WHS LETM1 + / - S4 growing either in presence of 25 mM glucose ( HG ) or BHB ( KB ) for 6 days . Scale bar 300 µm

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(C) Left panel: change in cell confluence of control (C1-C3), WHS LETM1+/- (S1-S4) and WHS LETM1+/+ (S5) fibroblasts grown in BHB over the course of 188 h. Data are expressed as means ± SEM of n=3 independent experiments. Right panel: images of control and WHS LETM1+/- S4 cells growing either in the presence of 25 mM glucose (HG) or BHB (KB) for 6 days. Scale bar 300 µm


( D ) Confocal of control ( C1 ) grown for 24 h in presence of BHB immunolabeled with anti - TOM20 ( red ) anti - ( green ) , DAPI stained ( blue )

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(D) Confocal images of control cells (C1) grown for 24 h in the presence of BHB and immunolabeled with anti-TOM20 (red) and anti-DNA (green), and DAPI stained (blue)


( E ) Proportion of that displayed mtDNA aggregation during KB treatment ( panel i - control ( C1 , C2 ) , WHS LETM1 + / + ( S5 ) fibroblasts ; panel ii - LETM1 + / - ( S1 , S2 , S4 ) fibroblasts ) . At least 50 per were counted at indicated time points . Data are expressed as mean ± of n = 3 independent experiments

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(E) Proportion of cells that displayed mtDNA aggregation during the KB treatment (panel i - control cells (C1, C2), and WHS LETM1+/+ (S5) fibroblasts; panel ii - LETM1+/- (S1, S2, S4) fibroblasts). At least 50 cells per cell line were counted at the indicated time points. Data are expressed as mean ± SEM of n=3 independent experiments


- seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated was prepared . Scatter plot showing log fold change ( LFC ) of GW7647 - upregulated genes ( LFC > 0 . 8 P < 0 . 05 ) in sham versus their LFC 10h after CLP . red represents diagonal , black represents real slope ( 0 . 3115 ) of data .

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RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data.


- seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated was prepared . of differentially expressed genes in sham mice after GW7647 treatment , involved in β - oxidation of fatty ( unit scale bar = log2 of normalized ) .

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RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts).


Confirmation of - seq data via qPCR on pure hepatocytes isolated via flow cytometry - sorting ( n = 3 / group ) . ( E ) Ppara ( F ) Hmgcs2 mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt Rpl . P - values were calculated using 2 - way ANOVA .

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Confirmation of RNA-seq data via qPCR on pure hepatocytes isolated via flow cytometry-based sorting (n = 3/group). (E) Ppara and (F) Hmgcs2 mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 2-way ANOVA analysis.


Mice ( n = 4 / group , data are representative of 2 experiments ) underwent a sham ( with or without starvation ) or CLP operation liver was isolated on several timepoints post - surgery for preparation qPCR . Ppara mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt Rpl . P - values were calculated using 1 - way ANOVA .

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Mice (n = 4/group, data are representative of 2 experiments) underwent a sham (with or without starvation) or CLP operation and liver was isolated on several timepoints post-surgery for RNA preparation and qPCR. Ppara mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis.


PPARA were analyzed in livers 24h after CLP by Western Blot using actin as a loading control .

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PPARA protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control.


of PPARA western blot . P - value was calculated via 2 - way Student ' s t - ( n = 4 / group ) .

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Quantification of PPARA western blot. P-value was calculated via 2-way Student's t-test (n = 4/group).


Pearson correlation between log fold change ( LFC ) Ppara expression 24h post - sepsis ( n = 38 , r = 0 . 6875 , combined data of 4 independent experiments ) .

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Pearson correlation between log fold change (LFC) Ppara expression levels and body temperature 24h post-sepsis (n= 38, r = 0.6875, combined data of 4 independent experiments).


Liver Hmgcs2 mRNA expression at different timepoints post - sepsis , expression is shown as relative expression , normalized to housekeeping genes Hprt Rpl . P - values were calculated using 1 - way ANOVA ( n = 4 / group , data are representative of 2 experiments ) .

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Liver Hmgcs2 mRNA expression at different timepoints post-sepsis, expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis (n = 4/group, data are representative of 2 experiments).


Oxygen consumption rates ( OCR ) of liver tissue explants 24h post sham or CLP . Liver tissue was isolated 24h post - surgery OCR was measured via Seahorse with BSA or palmitic acid ( PA ) as a substrate for 42 min . n = 4 / group .

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Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP. Liver tissue was isolated 24h post-surgery and OCR was measured via Seahorse with BSA or palmitic acid (PA) as a substrate for 42 min. n = 4/group.


Visualization of T42 ( min ) OCR , p - values were calculated using 2 - way ANOVA .

Visualization of T42(min) OCR, p-values were calculated using 2-way ANOVA analysis.


of that is taken up by inguinal fat pad ( iWAT , A ) , mesenteric fat pad ( mWAT , B ) perirenal fat pad ( pWAT , C ) 24h post - surgery in sham ( with or without starvation ) CLP mice . P - values were calculated with a 1 - way ANOVA . Combined data of 2 independent experiments , n = 6 / group .

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Percentage of body weight that is taken up by the inguinal fat pad (iWAT, A), the mesenteric fat pad (mWAT, B) and the perirenal fat pad (pWAT, C) 24h post-surgery in sham (with or without starvation) and CLP mice. P-values were calculated with a 1-way ANOVA test. Combined data of 2 independent experiments, n = 6/group.


Visual representation of ( D ) iWAT ( E ) pWAT sizes in sham , sham starved CLP mice 24h post - surgery . Data are representative of 3 independent experiments .

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Visual representation of (D) iWAT and (E) pWAT sizes in sham, sham starved and CLP mice 24h post-surgery. Data are representative of 3 independent experiments.


Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation blood was collected 6h 24h post - surgery . Plasma was isolated ( F ) FFA ( G ) glycerol was determined as described in method section . P - values were calculated with 2 - way ANOVA . Combined data of 2 independent experiments .

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Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Plasma was isolated and (F) FFA concentration and (G) glycerol concentration was determined as described in the method section. P-values were calculated with 2-way ANOVA tests. Combined data of 2 independent experiments.


Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation blood was collected 6h 24h post - surgery . Normalized abundances of ( H ) palmitic acid ( I ) palmitoyl - carnitine determined via liquid chromotography - mass spectrometric lipidomics . Values were normalized to IQ values , P - values were calculated with 2 - way ANOVA . n = 8 / group .

O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Normalized abundances of (H) palmitic acid and (I) palmitoyl-carnitine determined via liquid chromotography-mass spectrometric lipidomics. Values were normalized to IQ values , P-values were calculated with 2-way ANOVA tests. n = 8/group.


Immunofluorescent of liver kidney 24h after sham ( with or without starvation ) or CLP ( n = 6 - 7 / group , combined data of 2 independent experiments ) . scale bar = 20µm . Cryosections were stained with Acti - stain ( green ) , Hoechst ( blue ) , LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across entire tissue section .

O O O O O O O O O O O O O O O O O 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-MEASURED_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

Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section.


of lipid peroxidation by determination of ( C ) MDA ( D ) 4 - HNE concentrations in liver homogenates 24h post - surgery in sham CLP mice as described in methods ( n = 6 - 7 / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.


Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured by TUNEL , presented as % PI positive / µm² tissue . ( n = 6 / 7 mice / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA .

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

Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank . Combined data of 3 experiments , n = 20 / group .

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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 3 experiments, n = 20/group.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Liver samples were isolated 24h after CLP ( n = 5 - 7 / group , combined data of 2 independent experiments ) , mRNA was prepared gene expression of Ppara was analyzed via qPCR . Gene expression values are shown as relative expression , normalized to housekeeping genes Hprt Rpl . P - values were calculated via 2 - way ANOVA .

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 B-MEASURED_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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Liver samples were isolated 24h after CLP (n= 5-7/group, combined data of 2 independent experiments), mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR. Gene expression values are shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated via 2-way ANOVA test.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was isolated 24h after sepsis FFA ( D ) glycerol were determined as described in method section . P - values were calculated with 2 - way ANOVA . n = 5 - 7 / group , combined data of 2 independent experiments . Arrows represent % of decrease caused by pemafibrate treatment during sepsis .

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 B-MEASURED_VAR O O O B-MEASURED_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 B-CONTROLLED_VAR O O O O

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was isolated 24h after sepsis and FFA concentration and (D) glycerol concentration were determined as described in the method section. P-values were calculated with 2-way ANOVA tests. n = 5-7/group, combined data of 2 independent experiments. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Immunofluorescent of cryosections of liver 24h post - surgery that were stained with Acti - stain ( green ) , hoechst ( blue ) LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across entire tissue section . scale bar = 20µm .

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 B-MEASURED_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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Immunofluorescent images of cryosections of liver 24h post-surgery that were stained with Acti-stain (green), hoechst (blue) and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. White scale bar = 20µm.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . of lipid peroxidation by determination of MDA in liver homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA . Arrows represent % of decrease caused by pemafibrate treatment during sepsis .

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 B-MEASURED_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 B-CONTROLLED_VAR O O O O

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . of lipid peroxidation by determination of MDA in kidney homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA . Arrows represent % of decrease caused by pemafibrate treatment during sepsis .

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Oxygen consumption rates ( OCR ) of liver tissue explants 24h post CLP ( vehicle or pemafibrate treated ) after supplementation of palmitic acid ( PA ) , measured via Seahorse . Visualization of T42 ( min ) OCR . P - values were calculates using 2 - way ANOVA . One experiment , n = 3 / group .

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 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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Oxygen consumption rates (OCR) of liver tissue explants 24h post CLP (vehicle or pemafibrate treated) after supplementation of palmitic acid (PA), measured via Seahorse. Visualization of T42(min) OCR. P-values were calculates using 2-way ANOVA analysis. One experiment, n = 3/group.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery ( A ) Alanine - transferase ( ALT ) , were measured as described in methods . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and (A) Alanine-amino transferase (ALT), were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery creatinine concentrations , were measured as described in methods . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

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 B-MEASURED_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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and creatinine concentrations, were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery IL - 6 were measured as described in methods . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and IL-6 levels were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured with TUNEL depicted as % of PI positive / µm² tissue . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

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 O O O O O O

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in kidney paraffin - fixated sections 24h after sepsis , measured with TUNEL depicted as % of PI positive / µm² tissue . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Systemic load ( CFU / ml blood ) 24h post - sepsis in vehicle or pemafibrate treated mice . P - values were calculated with 2 - way Student ' s t - .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test.


Pemafibrate ( 1 mg / kg ) or vehicle ( 0 , 9 % NaCl ) was administered at different timepoints before after induction of sepsis survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via Log rank

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

Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank tests


Mice were injected with PPARα antagonist GW6471 ( 10µg / g ) or vehicle ( DMSO ) 3h pre - 3h post - CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank . Combined data of 2 independent experiments , n = 16 / group .

O O O O B-CONTROLLED_VAR 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

Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.


Blood samples were collected from healthy volunteers septic ( n = 13 septic , = 15 healthy controls ) . Plasma was prepared ( A ) FFA ( B ) glycerol concentrations were determined as described in methods . P - values were calculated with two - way Student t - .

O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Blood samples were collected from healthy volunteers and septic patients (n= 13 septic patients, = 15 healthy controls). Plasma was prepared and (A) FFA and (B) glycerol concentrations were determined as described in the methods. P-values were calculated with two-way Student t-tests.


C . cGAMP - VLPs induce an IFN - I response in target . HEK293 were infected with decreasing amounts of cGAMP - VLPs Empty - VLPs ( 1 / 5 serial dilutions starting at 2μL of VLP stocks per well ) infection was monitored 24 hours later by quantifying GFP + by flow cytometry . Supernatants from same infected were then transferred to a reporter expressing firefly luciferase under a promoter induced by IFN - I ( ISRE ) . Luciferase activity measured 24 hours later indicated presence of IFN - I in supernatants .

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

C. cGAMP-VLPs induce an IFN-I response in target cells. HEK293 cells were infected with decreasing amounts of cGAMP-VLPs and Empty-VLPs (1/5 serial dilutions starting at 2μL of VLP stocks per well) and the infection was monitored 24 hours later by quantifying GFP+ cells by flow cytometry. Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN-I (ISRE). Luciferase activity measured 24 hours later indicated the presence of IFN-I in the supernatants.


Immunisation with cGAMP - VLPs facilitates induction of HIV - 1 Gag - specific polyfunctional CD8 T responses . Alternatively , were analysed by ICS six hours after stimulation with peptide . CD8 T were gated as live , CD90 . 2 + , CD8 + . CD8 T expressing CD107a , IFNγ , TNFα or IL2 were analysed as shown in ( E ) .

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

Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E).


A , B . cGAMP loading enhances IgG responses specific to VLP , including VSV - G . ELISA plates were coated with lysate from cGAMP - VLPs ( A ) or recombinant VSV - G ( B ) . Antibodies of different isotypes specific for these were measured in sera from immunised mice by ELISA . optical density at increasing serum dilutions is shown in first three graphs from left EC50 is on right .

O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.


C . Enhanced antibody production following immunisation with cGAMP - VLPs relies on STING signalling . WT or Sting - / - mice were immunised with PBS , cGAMP - VLPs , Empty - VLPs or Empty - VLPs + poly ( I : C ) . IgG2b antibodies recognising VLP were assessed by ELISA . optical density at increasing serum dilutions is shown in first two graphs from left EC50 is on right .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR O O B-MEASURED_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

C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.


Immunisation with cGAMP - VLPs enhances accumulation of Tfh in draining lymph node . T follicular ( Tf ) were identified by flow cytometry as CD4 + CD44 + CXCR5hiPD1hi were further subdivided into Tfr ( FoxP3 + ) Tfh ( FoxP3 - ) . percentages of Tf , Tfh Tfr within CD4 + are shown in ( B ) .

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

Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).


Immunisation with VLPs induces germinal centre formation . Germinal centre B were identified by flow cytometry as B220 + IgD - CD95 + GL7 + . of germinal centre B amongst B220 + is shown in ( E ) .

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

Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E).