( 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 B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-DISEASE O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-DISEASE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-DISEASE 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD 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-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE 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-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD 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-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY 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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O

(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-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE 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 .

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) 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 .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O O O O

(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-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O 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-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY 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 .

O O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-CELL_LINE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(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-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY 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 ) .

O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O 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-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 . (

O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(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

O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O O 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-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O

(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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O

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 .

B-ORGANISM O O 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-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O

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 .

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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 .

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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. 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 .

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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-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O 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 .

B-EXP_ASSAY O B-TISSUE O O O O O O B-DISEASE O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O B-DISEASE B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-DISEASE 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-DISEASE 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-DISEASE 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 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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 .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-DISEASE O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O 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 - .

B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O B-DISEASE O O O B-SMALL_MOLECULE O B-ORGANISM 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-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-DISEASE B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-ORGANISM O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 - .

B-TISSUE O O O O O O B-DISEASE O O O O B-DISEASE O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O B-ORGANISM O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD 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-SMALL_MOLECULE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE B-CELL_TYPE O B-GENEPROD 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).


C , D . Low doses of cGAMP - HA - VLPs confer protection following IAV challenge . One month after immunisation with indicated doses of VLPs , were infected with 104 TCID50 of IAV PR8 . loss was monitored over following eleven days is shown as a of starting ( C , upper graph shows mean lower graphs show mice for each condition ) . approaching humane end - point of 20 % loss were culled survival to end - point curves are shown in ( D ) .

O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).


B . cGAMP - HA - VLPs Empty - HA - VLPs + AddaVax protect against IAV challenge . One month after immunisation , were infected with 104 TCID50 of IAV PR8 . approaching humane end - point of 20 % loss were culled survival to end - point curves are shown in ( B ) .

O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O

B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).


A . cGAMP - S - VLPs Empty - S - VLPs incorporate SARS - CoV - 2 S . Lysates from VLP producer VLP preparations were analysed by western blot for presence of S2 subunit of SARS - CoV - 2 S , Gag - GFP actin using indicated antibodies .

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A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.


B , C . Immunisation with cGAMP - S - VLPs augments anti - Spike anti - RBD antibody titres . Mice were immunised via intra - muscular route with 5x105 IU of cGAMP - S - VLPs , Empty - S - VLPs , or PBS as a control . Three after immunisation , sera were collected , heat - inactivated , titres of antibodies capable of binding to SARS - CoV - 2 S ( B ) or its RBD ( C ) were determined by ELISA . antibody response was expressed as Endpoint titre defined as reciprocal of highest serum dilution that gives a positive signal ( blank + 10SD ) . dotted shows limit of detection ( LOD ) .

O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O

B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).


D . Immunisation with cGAMP - S - VLPs induces neutralising antibodies . Using serum samples from ( B ) , antibody titres capable of neutralising SARS - CoV - 2 were determined by microneutralisation ( MN ) . Calculated IC50 doses from multiple serum dilutions are shown . dotted shows LOD .

O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O

D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.


A1 ) Representative WBs showing DRD2 RGS9 - 2 downregulation in adult ( P60 - P90 ) Tor1a + / - ( + / - ) striata , characterized by a reduced torsinA level with respect to Tor1a + / + ( + / + ) . striatal level of RGS7 is unchanged . dot plot shows Tor1a + / - data , normalized to Tor1a + / + controls of same experiment . TorsinA : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t * * * P = 0 . 0003 ; DRD2 : Tor1a + / + N = 18 , Tor1a + / - N = 16 , t * * P = 0 . 0029 ; RGS9 - 2 : Tor1a + / + N = 32 , Tor1a + / - N = 33 , t * P = 0 . 0115 ) . RGS7 : Tor1a + / + N = 7 , Tor1a + / - N = 7 , t P = 0 . 8721 ) . WB data , expressed as of vs . loading control level , are normalized to wild - type samples of same experiment . A2 , A3 , A4 ) Time - course of changes in torsinA , DRD2 RGS9 - 2 striatal along postnatal development . Summary plot data are normalized to Tor1a + / + P60 sample of each independent experiment . A2 ) TorsinA level is significantly reduced in mutants throughout developmental period considered ( P7 N = 3 ; P14 - P60 N = 4 ; one - way ANOVA P < 0 . 0001 Bonferroni ' s Multiple Comparison * * P < 0 . 01 at P7 , P14 P60 ) . A3 , A4 ) DRD2 RGS9 - 2 show parallel courses , with a similar increase from P7 to P21 in Tor1a + / + as well as Tor1a + / - striatal lysates ( P7 : DRD2 N = 3 , RGS9 - 2 N = 4 ; P14 - P21 N = 4 ; one - way ANOVA with Bonferroni ' s Multiple Comparison P > 0 . 05 ) . At P60 , a simultaneous reduction of DRD2 RGS9 - 2 is observed in Tor1a + / - striatal lysates ( N = 4 ; Tor1a + / - one sample t DRD2 : * P = 0 . 0250 , RGS9 - 2 : * P = 0 . 0175 ) . WB data , expressed as of vs . loading control level , are normalized to wild - type P60 sample of same experiment

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment


B ) Representative WB dot plot show downregulation of torsinA , DRD2 RGS9 - 2 striatal also in adult P60 - P90 Tor1a ∆ gag / + ( ∆ gag / + ) mice . TorsinA : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 12 , t * * * P = 0 . 0006 ; DRD2 : Tor1a + / + N = 7 , Tor1a ∆ gag / + N = 9 , t * P = 0 . 0183 ; RGS9 - 2 : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 9 , t * P = 0 . 0192 . WB data , expressed as of vs . loading control level , are normalized to wild - type samples of same experiment . Mean ± is represented in

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O 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) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphs


C ) Left . Representative image of coronal striatal slices of fresh frozen brains of wild - type Tor1a + / - mice probed with DRD2 radioligand 3H - spiperone . Scale bar 1 . 5 mm . ( Right ) Graph showing DRD2 binding density in Tor1a + / - striatal sections , obtained from densitometric , expressed as variation compared to control

O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O

C) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals


A1 ) Representative WB of RGS9 - 2 , R7BP , Gβ5 on four Tor1a + / + four Tor1a + / - striatal lysates . bar histogram on right shows of three in each sample . A2 ) Dot plot showing that Gβ5 striatal are unchanged in Tor1a + / - mice ( Tor1a + / + N = 11 , Tor1a + / - N = 10 , Mann Whitney P = 0 . 2453 ) . A3 ) Conversely , level of R7 binding R7BP is increased in Tor1a + / - striatum ( Tor1a + / + N = 12 , Tor1a + / - N = 12 , t * * P = 0 . 0014 ) . Values are represented as of vs . loading control level , normalized to mean of Tor1a + / + control values of same

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD 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-GENEPROD O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O

A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment


B1 ) Representative WB of lysates of Tor1a + / + Tor1a + / - dorsal striatum slices incubated for 5 hours in presence ( w / leu ) or absence ( w / o leu ; striatum ) of protease inhibitor leupeptin . Samples showing enhanced degradation of DRD2 / or RGS9 - 2 show additional bands at lower molecular . B2 ) Summary plot reporting mean ± of RGS9 - 2 DRD2 level values , expressed as of vs . loading control level , normalized to value of Tor1a + / + w / leu sample measured in same experiment . DRD2 : Tor1a + / + N = 4 , t P = 0 . 4376 ; Tor1a + / - N = 4 , t * P = 0 . 0427 ; RGS9 - 2 : Tor1a + / + N = 5 , t * P = 0 . 0464 ; Tor1a + / - N = 5 , t * P = 0 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190


C1 , C2 , C3 , C4 ) Dot plots showing DRD2 , RGS9 - 2 , R7BP Gβ5 measured in striatal detergent - resistant - ( DRM ) preparations from Tor1a + / - ( + / - ) WT ( + / + ) mice ( DRD2 : Tor1a + / + N = 16 , Tor1a + / - N = 13 , t P = 0 . 0129 ; RGS9 - 2 : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t * P = 0 . 0396 ; R7BP : Tor1a + / + N = 6 , Tor1a + / - N = 6 , t * * * P = 0 . 0002 ; Gβ5 : Tor1a + / + N = 9 , Tor1a + / - N = 9 , t P = 0 . 8461 )

O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)


WB of markers of endocytic trafficking degradative pathway in dorsal striatum of Tor1a + / - mice . A ) level of β - arrestin 2 ( β - Arr2 ) is not altered ( Tor1a + / + N = 11 , Tor1a + / - N = 12 , t P = 0 . 6796 ) , whereas level of spinophilin ( B ) is decreased ( Tor1a + / + N = 5 , Tor1a + / - N = 8 , t * P = 0 . 0151 )

B-EXP_ASSAY O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151)


C ) A Sh - RGS9 - 2 - GFP viral preparation injected into right striatum ( + ) of C57Bl / 6 mice effectively reduces both RGS9 - 2 DRD2 , with respect to striatum ( - ) . summary plot reports mean ± values ( RGS9 - 2 : N = 4 , paired t * P = 0 . 0100 ; DRD2 : N = 4 , paired t * P = 0 . 0432 )

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

C) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432)


D ) endosomal marker Rab4 is upregulated in Tor1a + / - striatum ( Tor1a + / + N = 13 , Tor1a + / - N = 14 , t * * P = 0 . 0087 )

O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

D) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087)


E ) Basal of markers of degradative pathway , p62 LC3 - II , show a trend towards upregulation in Tor1a + / - striatal lysates ( p62 : Tor1a + / + N = 11 , Tor1a + / - N = 11 , t P = 0 . 2292 ; LC3 - II : Tor1a + / - N = 9 , Tor1a + / - N = 11 , t P = 0 . 1574 )

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

E) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574)


F ) Treatment with bafilomycin A1 ( BafA1 ) induces a significantly more pronounced increase of LC3 - II in Tor1a + / - than in wild - type dorsal striatum slices . BafA1 - induced increase in LC3 - II level measured in Tor1a + / - slices was normalized to increase observed in Tor1a + / + samples of same experiment ( N = 4 , one sample t * * P = 0 . 0012 )

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)


G ) LAMP - 2 , a lysosomal marker , shows a trend for upregulation ( Tor1a + / + N = 8 ; Tor1a + / - N = 6 , t P = 0 . 2232 )

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G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232)


Representative double immunofluorescence confocal of coronal sections of dorsal striatum showing p62 immunolabelling ( red ) in choline acetyltransferase ( ChAT ) - positive ( cyano ) ChIs of Tor1a + / + Tor1a + / - mice . A1 ) Two representative ChIs identified by box in low magnification ( bar = 20 µm ) are shown at higher magnification in ( A2 ) ( bar = 5 µm ) . A3 ) graph reporting mean ± values of p62 / ChAT signal overlap in ChIs , normalized to Tor1a + / + values of same experiment , shows a significant increase of p62 in Tor1a + / - neurons ( Tor1a + / + n = 20 , N = 6 mice ; Tor1a + / - n = 20 , N = 6 mice , t * * * P = 0 . 00008 )

O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-TISSUE O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008)


A ) Top . Low magnification merged confocal of coronal striatal sections showing ChAT - labelled ( cyano ) ChIs in of mCherry ( red ) indicating viral infection . Bar = 100 µm . Bottom . Higher magnification of ChIs identified by box in upper show viral - induced expression of mCherry GFP signal . Bar = 20 µm . B ) mCherry / ChAT overlap is increased in ChIs from Tor1a + / - mice ( Tor1a + / + n = 24 , Tor1a + / - n = 22 , N = 8 mice / genotype , t * * * P = 0 . 0005 ) , but mCherry / GFP indicated that LC3 flux was unchanged ( Tor1a + / + n = 19 , Tor1a + / - n = 21 , N = 8 mice / genotype , Mann Whitney P = 0 . 2334 )

O O O O O O O B-EXP_ASSAY O O B-TISSUE O O B-GENEPROD O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)


C ) Merged split channels confocal ( bar = 10 µm ) of representative Tor1a + / + Tor1a + / - ChIs showing red dots yellow ( indicated by circles ) , quantified in ( D ) : red dots : Tor1a + / + n = 13 , Tor1a + / - n = 14 , t * * * P = 0 . 0003 ; yellow dots : Tor1a + / + n = 13 , Tor1a + / - n = 14 , t * * P = 0 . 0048 , N = 5 mice / genotype ) . Measurement of mCherry / yellow dots indicated that autophagic flux was not altered ( Tor1a + / + n = 13 ; Tor1a + / - n = 14 , Mann Whitney P = 0 . 8842 ) . Data in B , D ) are represented as mean ±

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O 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) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM