EMBO/SourceData huggingface dataset:
Figure 4(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.
Figure 5(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 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).
Figure 3(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 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.(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) 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).
Figure 4(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 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.
Figure 5(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 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 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) Quantification result of (E). Data are presented as mean± SD, n = 8 technically independent samples.(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 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).
Figure 6(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 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) 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 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) 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) 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).
Figure 7(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 and C, result of rapamycin treatment). Mean± SD; n= 5 technically independent samples.(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 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.
Figure 1 (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 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 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) 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
Figure 3 (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 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 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
Figure 1RNA-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.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 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.
Figure 2Mice (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 protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control.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 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 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 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 analysis.
Figure 3Percentage 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 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 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 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.
Figure 4Immunofluorescent 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.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 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.
Figure 5Mice 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 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 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 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. 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. 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 analysis. One experiment, n = 3/group.
Figure 6Mice 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 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 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 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 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 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 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 testsMice 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.
Figure 7Blood 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.
Figure 1C. 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.