( G ) Representative western blot of immunoprecipitated ( IP ) HTT ( ab - MAB2166 ) HTT pS13 ( ab - CHDI - 90001039 - 1 ) after lentivirus ( LV ) - mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat striatal neuronal . ( H ) of fold changes in HTT pS13 to total HTT compared to those of kinase - dead mutant from experiments like in G ( n = 3 ) .

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

(G) Representative western blot of immunoprecipitated (IP) HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) after lentivirus (LV)-mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells. (H) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in G (n=3).


( A ) Representative western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with indicated kinases for 48 hour in HEK293T . ( B ) graph indicates fold change in HTT in samples like illustrated in A , relative to empty vector controls normalized to GAPDH .

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

(A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (B) The graph indicates the fold change in HTT in samples like illustrated in A, relative to empty vector controls normalized to GAPDH.


( C ) Representative immunoblot of HTT ( ab - MAB5492 ) after filter retardation from insoluble fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with indicated kinases for 48 hour in HEK293T . ( D ) of fold change in HTT compared to EV expressing HTTex1 16Q from experiments like in C .

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

(C) Representative immunoblot of the HTT (ab-MAB5492) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (D) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C.


( E ) Representative immunofluorescence of HTT ( eGFP ) TBK1 ( ab - anti - MYC ) upon coexpression of HTTex1 72Q eGFP or its phosphorylation - incompetent mutants with indicated kinases for 48 hour in HEK293T ( scale bar 20μm ) .

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

(E) Representative immunofluorescence images of HTT aggregates (eGFP) and TBK1 (ab-anti-MYC) upon coexpression of HTTex1 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells (scale bar 20μm).


( F ) of of co - transfected presenting upon expression of indicated kinase , from experiments like in E . ( G ) of of presenting from experiments like in E .

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

(F) Quantification of the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in E. (G) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E.


( H ) Representative western blots of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) in soluble fraction from HEK293T transfected first with HTTex1 72Q eGFP then after 48 hour ( to enable formation of before kinase expression ) ; with TBK1 ; were lysed at indicated time after kinase transfection . ( I ) of of presenting kinase expression from experiments in H .

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

(H) Representative western blots of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour (to enable the formation of aggregates before the kinase expression); with TBK1; cells were lysed at the indicated time after kinase transfection. (I) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H.


( A of by a condensation , were fixed nuclei were stained with Hoechst dye , with a higher than average plus two standard deviations are considered dead . ( A ) of at DIV14 , after co - transfection of indicated kinases HTTex1 plasmid in rat striatal neurons at DIV9 ( n = 3 ) ,

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

(A Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (A) The percentage of cell death at DIV14, after co-transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 (n=3),


B ) of by a condensation , were fixed nuclei were stained with Hoechst dye , with a higher than average plus two standard deviations are considered dead . ( B ) of at DIV7 , after co - transfection of indicated kinases HTT N586 plasmid in mouse striatal neurons at DIV5 ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (B) The percentage of cell death at DIV7, after co-transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 (n=8).


( C ) of by a TUNEL in rat striatal neurons transfected with indicated kinases at DIV9 with indicated HTT plasmid . were fixed at DIV14 , of TUNEL - positive neurons was counted plotted ( n = 3 ) .

O O O O O O O O O O O O O O O O O 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) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed at DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).


( D ) of . , by a condensation in mouse cortical neurons co - transfected with indicated HTT N586 kinases at DIV5 , were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7 . were fixed , nuclei were stained with Hoechst dye ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 percentage of cell death., quantification by a nuclear condensation assay in mouse primary cortical neurons co-transfected with the indicated HTT N586 and kinases at DIV5, cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7. Cells were fixed, and the nuclei were stained with Hoechst dye (n=3).


( E ) of by a TUNEL in mouse striatal neurons transfected with indicated kinases at DIV9 with indicated HTT plasmid . were fixed on DIV14 , of TUNEL - positive neurons was counted plotted ( n = 3 ) .

(E) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed on DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).


( A ) Representative immunofluorescence image of C . elegans overexpressing HTT N513 Q15 HTT N513 Q128 ( scale bar 20μm ) . Top panel complete worm , lower panel head region .

(A) Representative immunofluorescence image of C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 (scale bar 20μm). Top panel complete worm, lower panel head region.


( B ) Western blot showing HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) HTT pT3 ( ab - CHDI - 90001528 - 1 ) in C . elegans overexpressing HTT N513 Q15 HTT N513 Q128 . ( C ) of fold changes in ratios of HTT pS13 pT3 to total HTT in C . elegans overexpressing HTT N513 Q15 HTT N513 Q128 from experiments like in B ( n = 3 ) .

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

(B) Western blot showing HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) levels in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128. (C) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B (n=3).


( D ) Representative western blots showing HTT ( ab - MAB5492 ) HTT pS13 ( ab - CHDI - 90001039 - 1 ) from detergent soluble fraction HTT ( ab - MAB5492 ) from detergent insoluble fraction in three different transgenic C . elegans lines overexpressing TBK1 or TBK1 KD . ( E ) bottom graph indicates fold change in HTT pS13 ( ab - CHDI - 90001039 - 1 ) ratios to total HTT ( ab - MAB5492 ) compared to those in kinase - dead mutant 1 from experiments like in D . top graph indicates fold change in HTT ( ab - MAB5492 ) compared to that in kinase - dead mutant experiments like in D ( n = 3 ) .

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

(D) Representative western blots showing the HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) levels from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in three different transgenic C. elegans lines overexpressing TBK1 or the TBK1 KD. (E) The bottom graph indicates the fold change in the HTT pS13 (ab-CHDI-90001039-1) ratios to total HTT (ab-MAB5492) compared to those in the kinase-dead mutant line 1 from the experiments like in D. The top graph indicates the fold change in HTT aggregates (ab-MAB5492) compared to that in the kinase-dead mutant the experiments like in D (n=3).


( F ) Mobility of Q15 Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 26 - 29 ) .

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

(F) Mobility of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n= 26-29).


( G ) Survival graph of Q15 Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 30 ) .

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

(G) Survival graph of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n=30).


( H ) Representative western blots showing HTT ( ab - MAB5492 ) HTT pS13 ( ab - CHDI - 90001039 - 1 ) from detergent soluble fraction HTT ( ab - MAB5492 ) from detergent insoluble fraction in TBK1 orthologue ( ikke - 1 or unc - 51 ) RNAi - treated C . elegans lines expressing HTT N513 Q15 HTT N513 Q128 . ( I ) of fold change in of pS13 HTT to total HTT compared to non - targeting RNAi from experiments like in H ( n = 4 ) .

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

(H) Representative western blots showing HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in TBK1 orthologue (ikke-1 or unc-51) RNAi-treated C. elegans lines expressing HTT N513 Q15 and HTT N513 Q128. (I) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non-targeting RNAi from the experiments like in H (n=4).


( A ) Representative immunoblot of a filter retardation of HTT ( ab - MAB5492 ) from insoluble fraction ; HEK293T co - expressed HTTex1 72Q eGFP TBK1 or TBK1 KD for 48 hour , for last 16 hour , they were treated with indicated proteasome inhibitor ( MG132 , 5 µM ) or autophagy inhibitor ( Baf A1 , 200 nM , NH4Cl , 10 mM , 3 - MA , 5 mM ) . ( B ) of fold change in HTT compared to TBK1 KD treated with DMSO from blots like in A ( n = 3 ) .

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

(A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM). (B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3).


( C ) Immunoblot of LC3 ( ab48394 ) from soluble HEK293 fractions overexpressing TBK1 or TBK1 KD for 24 hour ; for last hour , Baf A1 ( 500 nM ) was added as indicated . ( D ) Fold change in LC3 - II ( lower band ) relative to respective untreated kinase dead mutant normalized to actin from blots like in C ( n = 3 ) .

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

(C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated. (D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3).


( E ) Immunoblot of LC3 ( ab48394 ) from soluble rat neuronal overexpressing lentivirus - mediated TBK1 TBK1 KD for 96 hour . For last 1 or 4 hour , Baf A1 ( 500 nM ) was added as indicated . ( F ) of fold change in LC3 - II ( lower band ) compared to kinase dead mutant , which was untreated , normalized to GAPDH from blots like in E ( n = 3 ) .

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

(E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated. (F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).


( A ) Western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) HTT ( ab - MAB5492 ) , upon coexpression of HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A / S16A variant with TBK1 KD , TBK1 or TBK1 Δ690 - 713 for 48 hour in HEK293 . ( B ) graph indicates calculated fold changes in HTT , HTT pS13 to total HTT HTT compared to in TBK1 KD from experiments like in A ( bottom panel normalized to Tubulin , n = 3 ) .

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

(A) Western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT aggregates (ab-MAB5492), upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A/S16A variant with TBK1 KD, TBK1 or TBK1 Δ690-713 for 48 hour in HEK293 cells. (B) The graph indicates the calculated fold changes in HTT, HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A (bottom panel normalized to Tubulin, n=3).


( C ) Representative immunofluorescence of eGFP HTTex1 72Q or its phosphorylation - deficient variant S13A / S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690 - 713 ( ab - anti - Myc ) with for 48 hour in HEK293 ( scale bar 20μm ) . ( D ) graph indicates of co - transfected presenting upon expression of indicated kinase , from experiments like in C ( n = 3 ) .

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

(C) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation-deficient variant S13A/S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690-713 (ab-anti-Myc) with for 48 hour in HEK293 cells (scale bar 20μm). (D) The graph indicates the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in C (n=3).


A - O ( A , F , K ) slices through electron cryo - tomograms of L . pneumophila , P . aeruginosa , S . oneidensis , respectively , highlighting a PL sub - in outer ( dashed yellow circle ) . ( B , G , L ) Enlarged views of complexes . ( C , H , M ) Sub - tomogram averages of PL sub - complexes from each species . ( D , I , N ) Schematic representations of sub - tomogram averages with different rings colored labeled . ( E , J , O ) Sub - tomogram averages of fully - assembled flagella from each species for comparison . Scale bars : ( black ) 50 nm , ( orange ) 25 nm , ( red ) 20 nm .

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

A-O (A, F, K) slices through electron cryo-tomograms of L. pneumophila, P. aeruginosa, and S. oneidensis cells, respectively, highlighting a PL sub-complex in the outer membrane (dashed yellow circle). (B, G, L) Enlarged views of the complexes. (C, H, M) Sub-tomogram averages of PL sub-complexes from each species. (D, I, N) Schematic representations of the sub-tomogram averages with different rings colored and labeled. (E, J, O) Sub-tomogram averages of fully-assembled flagella from each species for comparison. Scale bars: (black) 50 nm, (orange) 25 nm, (red) 20 nm.


Sub - tomogram average of IM sub - constituting C - ring , MS - ring export apparatus . Schematic representation of sub - tomogram average shown in ( A ) highlighting different parts of . Sub - tomogram average of motors of fully - assembled flagella . Slices through electron cryo - tomograms showing neighboring PL IM sub - complexes . Dashed - yellow circles highlight IM sub - while dashed - blue arrows highlight PL sub - . Dashed - red lines mark border between two used to make a composite image when PL IM sub - complexes were found at different in tomogram . Schematic representation of an IM sub - in vicinity of a PL sub - . Slices through electron cryo - tomograms of L . pneumophila highlighting presence of a fully - assembled basal lacking hook filament . Schematic representation of basal bodies in ( L M ) . Central slice through an electron cryo - tomogram of a lysed . dashed - yellow circle highlights flagellar motor . Enlarged view of same slice shown in ( O ) . absence of C - ring export apparatus is highlighted by dashed - orange arrow . Schematic representation of found in ( P ) .

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

Sub-tomogram average of the IM sub-complex constituting the C- ring, MS-ring and export apparatus. Schematic representation of the sub-tomogram average shown in (A) highlighting the different parts of the complex. Sub-tomogram average of the motors of fully-assembled flagella. Slices through electron cryo-tomograms showing neighboring PL and IM sub-complexes. Dashed-yellow circles highlight the IM sub-complex while dashed-blue arrows highlight the PL sub-complex. Dashed-red lines mark the border between two images used to make a composite image when the PL and IM sub-complexes were found at different levels in the tomogram. Schematic representation of an IM sub-complex in the vicinity of a PL sub-complex. Slices through electron cryo-tomograms of L. pneumophila highlighting the presence of a fully-assembled basal body lacking the hook and the filament. Schematic representation of the basal bodies in (L and M). Central slice through an electron cryo-tomogram of a lysed cell. The dashed-yellow circle highlights the flagellar motor. Enlarged view of the same slice shown in (O). The absence of the C-ring and the export apparatus is highlighted by the dashed-orange arrow. Schematic representation of the complex found in (P).


Slices through electron cryo - tomograms showing fully - assembled motors without hook filament . dashed - yellow circles indicate IM sub - . Schematic representation of P . aeruginosa motors lacking hook filament shown in ( A - C ) . Slices through electron cryo - tomograms showing fully - assembled motors with hook lacking filament . Schematic representation of motors with hook shown in ( E - G ) . Slices through electron cryo - tomograms of intact P . aeruginosa ∆ flgI showing presence of inner - sub - with rod . Schematic representation of inner - sub - with rod shown in ( I - K ) . Slices through electron cryo - tomogram of lysed P . aeruginosa ∆ flgI showing presence of sub - constituting MS - ring rod . Schematic representation of sub - described in ( M - O ) . A slice through an electron cryo - tomogram of a P . aeruginosa ∆ flgG highlighting presence of inner - sub - . Schematic representation of inner - sub - shown in ( Q ) .

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

Slices through electron cryo-tomograms showing fully-assembled motors without the hook and filament. The dashed-yellow circles indicate the IM sub-complex. Schematic representation of the P. aeruginosa motors lacking the hook and filament shown in (A-C). Slices through electron cryo-tomograms showing fully-assembled motors with the hook and lacking the filament. Schematic representation of the motors with the hook shown in (E-G). Slices through electron cryo-tomograms of intact P. aeruginosa ∆flgI cells showing the presence of the inner-membrane sub-complex with the rod. Schematic representation of the inner-membrane sub-complex with the rod shown in (I-K). Slices through electron cryo-tomogram of lysed P. aeruginosa ∆flgI cells showing the presence of the sub-complex constituting the MS-ring and the rod. Schematic representation of the sub-complex described in (M-O). A slice through an electron cryo-tomogram of a P. aeruginosa ∆flgG cell highlighting the presence of the inner-membrane sub-complex. Schematic representation of the inner-membrane sub-complex shown in (Q).


Slices through electron cryo - tomograms of wild type showing fully - assembled motors without hook filament . Schematic representation of motors lacking hook filament shown in ( A - C ) . Sub - tomogram average of motors of fully - assembled flagella . Slice through an electron cryo - tomogram of a ΔflgH showing an IM sub - , indicated by dashed - yellow circle . Schematic representation of IM sub - shown in ( F ) . Slices through electron cryo - tomograms of ΔflgH showing IM sub - with rod P - ring , indicated by dashed - yellow circles . Schematic representation of structures shown in ( H I ) . Slices through electron cryo - tomograms of ΔflaA / B highlighting flagellar motor hook ( without filament ) . Sub - tomogram average of flagellar motor hook structure found in ΔflaA / B . Schematic representation of sub - tomogram average shown in ( N ) . Slices through electron cryo - tomograms of ΔflaA / B illustrating a disassembly product constituting PL sub - , rod hook . Sub - tomogram average of disassembly shown in ( P - R ) . dashed - orange arrow indicates absence of IM sub - in this structure . Schematic representation of disassembly product shown in ( P - S ) . Slices through electron cryo - tomograms of ΔflaA / B highlighting PL sub - complexes ( dashed - yellow circles ) . Sub - tomogram average of PL sub - complexes in ΔflaA / B . Schematic representation of sub - tomogram average shown in ( X ) .

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

Slices through electron cryo-tomograms of wild type cells showing fully-assembled motors without the hook and filament. Schematic representation of the motors lacking the hook and filament shown in (A-C). Sub-tomogram average of the motors of fully-assembled flagella. Slice through an electron cryo-tomogram of a ΔflgH cell showing an IM sub-complex, indicated by the dashed-yellow circle. Schematic representation of the IM sub-complex shown in (F). Slices through electron cryo-tomograms of ΔflgH cells showing the IM sub-complex with the rod and the P-ring, indicated by the dashed-yellow circles. Schematic representation of the structures shown in (H and I). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting the flagellar motor and the hook (without the filament). Sub-tomogram average of the flagellar motor and the hook structure found in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (N). Slices through electron cryo-tomograms of ΔflaA/B cells illustrating a disassembly product constituting the PL sub-complex, the rod and the hook. Sub-tomogram average of the disassembly complex shown in (P-R). The dashed-orange arrow indicates the absence of the IM sub-complex in this structure. Schematic representation of the disassembly product shown in (P-S). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting PL sub-complexes (dashed-yellow circles). Sub-tomogram average of the PL sub-complexes in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (X).


A , B . Immunoblotting were performed to validate Mettl3 or Mettl14 expression in CT26 B16 as indicated . Gapdh served as a loading control .

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A, B. Immunoblotting were performed to validate Mettl3 or Mettl14 expression levels in CT26 and B16 cells as indicated. Gapdh served as a loading control.


volume was monitored for control Mettl3 or Mettl14 - depleted tumors with treatment as indicated in CT26 colon cancer Data are mean ± s . e . m of indicated number of mice in each group . n , numbers of mice . * P < 0 . 05 ; * * * P < 0 . 001 by Student ' s t - .

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Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in CT26 colon cancer Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests.


volume was monitored for control Mettl3 or Mettl14 - depleted tumors with treatment as indicated in B16 melanoma , respectively . Data are mean ± s . e . m of indicated number of mice in each group . n , numbers of mice . * P < 0 . 05 ; * * * P < 0 . 001 by Student ' s t - .

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Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in B16 melanoma, respectively. Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests.


E , F . Survival of control tumors those with depleted genes were recorded as indicated in CT26 colon cancer B16 melanoma , respectively . Data are mean ± s . e . m . of indicated number of mice in each group . n , numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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

E, F. Survival analysis of control tumors and those with depleted genes were recorded as indicated in CT26 colon cancer and B16 melanoma, respectively. Data are mean ± s.e.m. of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


A . of - infiltrating T , Treg NK were identified by flow cytometry from CT26 tumors as indicated . Each spot represents one mouse . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - .

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A. Percentage of tumor-infiltrating T cells, Treg and NK cells were identified by flow cytometry from CT26 tumors as indicated. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests.


B . Representative of CD8 by . Tissue sections from BALB / c mice bearing indicated knockout of genes with treatment of PD1 antibody . Scale bars , 50 μm .

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B. Representative images of CD8 by IHC staining. Tissue sections from BALB/c mice bearing the indicated knockout of genes with treatment of PD1 antibody. Scale bars, 50 μm.


C . of Granzyme B - expressing CD8 + T from control Mettl3 or Mettl14 deficient CT26 tumors . Each spot represents one mouse . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - .

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C. Percentage of Granzyme B-expressing CD8+ T cells from control and Mettl3 or Mettl14 deficient CT26 tumors. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests.


Mice bearing control Mettl3 or Mettl14 null tumors were treated with CD8 - depleting antibody PD - 1 antibody or PD - 1 as indicated . volume was measured over time points . n , numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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

Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1 as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


Mice bearing control Mettl3 or Mettl14 null tumors were treated with CD8 - depleting antibody PD - 1 antibody or PD - 1 / GVAX as indicated . volume was measured over time points . n , numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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

Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1/GVAX as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


IFNγ production in serum ( F ) from BALB / c mice by ELISA . results are representatives of at least three independent experiments . n , numbers of mice . Data are mean ± s . e . m . * P < 0 . 05 by Student ' s t - .

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

IFNγ production in serum (F) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests.


IFNγ production in Intratumor ( G ) from BALB / c mice by ELISA . results are representatives of at least three independent experiments . n , numbers of mice . Data are mean ± s . e . m . * P < 0 . 05 by Student ' s t - .

IFNγ production in Intratumor (G) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests.


A . Volcano plot of differentially expressed genes obtained by DESeq2 in Mettl3 or Mettl14 null tumors compared to control tumors . Significantly upregulated or downregulated genes are plotted in red blue points , respectively . n . s , non - significant .

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A. Volcano plot of differentially expressed genes obtained by DESeq2 analysis in Mettl3 or Mettl14 null tumors compared to control tumors. Significantly upregulated or downregulated genes are plotted in red and blue points, respectively. n.s, non-significant.


F . Representative genes with m6A sites generated by integrative genomics viewer . Data is representative of duplicates with similar results . Red represents reads coverage of IP sample blue represents reads coverage of input sample . Rectangular cyan shade represents m6A peaks located on transcripts .

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F. Representative genes with m6A sites generated by integrative genomics viewer. Data is representative of duplicates with similar results. Red represents reads coverage of IP sample and blue represents reads coverage of input sample. Rectangular cyan shade represents the m6A peaks located on transcripts.


G . m6A enrichment of Stat1 Irf1 was examined by m6A RIP - qPCR in control , Mettl3 , or Mettl14 depleted CT26 tumors as indicated . Ctla4 functioned as a m6A negative control ( Wang et al . , 2019 ) Data are mean ± s . d . * * P < 0 . 01 by Student ' s t - .

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

G. m6A enrichment of Stat1 and Irf1 was examined by m6A RIP-qPCR in control, Mettl3, or Mettl14 depleted CT26 tumors as indicated. Ctla4 functioned as a m6A negative control (Wang et al., 2019) Data are mean ± s.d. * * P < 0.01 by Student's t-tests.


H . Immunoblots of p - Stat1 ( phosphorylated ) , Stat1 Irf1 were carried out in indicated tumors in triplicate with Gapdh as a loading control .

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

H. Immunoblots of p-Stat1 (phosphorylated), Stat1 and Irf1 were carried out in the indicated tumors in triplicate with Gapdh as a loading control.


I . growth from CT26 with Mettl3 , Mettl14 , Mettl3 / Stat1 , Mettl3 / Irf1 , Mettl14 / Stat1 or Mettl14 / Irf1 - depleted genes control under treatment of PD - 1 antibody as indicated . n , numbers of mice . Data are mean ± s . e . m of indicated number of mice in each group .

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

I. Tumor growth from CT26 cells with Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1 or Mettl14/Irf1-depleted genes and control under treatment of PD-1 antibody as indicated. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice in each group.


A . proliferation of Mettl3 or Mettl14 depleted control CT26 treated with indicated combinations of cytokines for 48h . mean ± SD of three replicates is shown . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - .

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A. Cellular proliferation analysis of Mettl3 or Mettl14 depleted and control CT26 cells treated with indicated combinations of cytokines for 48h. The mean ± SD of three replicates is shown. * P < 0.05; * * P < 0.01 by Student's t-tests.


B . BALB / c mice bearing Mettl3 or Mettl14 deficient control tumors were treated with IFNγ - blocking antibody PD - 1 antibody as indicated . was measured over time . n , numbers of mice . Data are mean ± s . e . m of indicated number of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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

B. BALB/c mice bearing Mettl3 or Mettl14 deficient and control tumors were treated with IFNγ -blocking antibody and PD-1 antibody as indicated. Tumor size was measured over time. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


C . Quantitative RT - PCR was performed to identify transcriptional changes of IFN - γ response gene expression ( n = 3 ) . Data are shown as relative fold change ( RFC , color coded bar ) .

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C. Quantitative RT-PCR was performed to identify transcriptional changes of the IFN-γ response gene expression (n=3). Data are shown as the relative fold change (RFC, color coded bar).


D , E . mRNA stability of Stat1 Irf1 were measured by qRT - PCR in treated with IFN - γ actinomycin D . Mean ± SD of n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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

D, E. mRNA stability of Stat1 and Irf1 were measured by qRT-PCR in tumor cells treated with IFN-γ and actinomycin D. Mean ± SD of n=3. * * P < 0.01; * * * P < 0.001 by Student's t-tests.


F . Validation effect of knock out of Ythdf1 - 3 using western blotting , Gapdh served as a loading control .

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F. Validation the effect of knock out of Ythdf1-3 using western blotting, Gapdh served as a loading control.


G . qPCR of Stat1 Irf1 ' s expression in indicated depletion of CT26 with / without stimulation of IFNγ . Mean ± SD of n = 3 . * P < 0 . 05 by Student ' s t - .

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G. qPCR analysis of Stat1 and Irf1's expression in the indicated depletion of CT26 cells with/without stimulation of IFNγ. Mean ± SD of n=3. * P < 0.05 by Student's t-tests.


H . Western blot of Mettl3 Mettl14 in overexpressed CT26 . Gapdh served as a loading control for each .

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H. Western blot analysis of Mettl3 and Mettl14 in overexpressed CT26 cells. Gapdh served as a loading control for each.


I , J . qPCR of mRNA stability of Stat1 Irf1 in indicated CT26 treated with IFNγ actinomycin D . Mean ± SD of n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - .

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I, J. qPCR analysis of the mRNA stability of Stat1 and Irf1 in the indicated CT26 cells treated with IFNγ and actinomycin D. Mean ± SD of n=3. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


A , B . level of STAT1 was negatively correlated with METTL3 METTL14 in human pMMR - MSI - L CRC colon tissue ( r2 = - 3 . 2477 for METTL3 , r2 = - 2 . 7491 for METTL14 ) . Each dot represents one tissue .

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A, B. The protein level of STAT1 was negatively correlated with METTL3 and METTL14 in human pMMR-MSI-L CRC colon tissue (r2=-3.2477 for METTL3, r2=-2.7491 for METTL14). Each dot represents one tumor tissue.


A . Detection of Sufu Skp1 after immunoprecipitation of indicated Flag - tagged F - Box ( FBPs ) . An Empty Vector ( EV ) was used as a negative control . HEK293T were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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A. Detection of Sufu and Skp1 after immunoprecipitation of the indicated Flag-tagged F-Box Proteins (FBPs). An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


B , C . Immunoprecipitation of endogenous Sufu from PC3 ( B ) DAOY ( C ) transfected with either an empty vector ( EV ) or Myc - tagged Fbxl17 . Non - specific rabbit immunoglobulin G ( IgG ) was used as a negative control . Detection of chains of immunoglobulin ( IgG ) was used to assess amount of IgG used for each immunoprecipitation reaction . Treatment with MLN4924 ( 2 M ) was started 5 hours before collection .

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B, C. Immunoprecipitation of endogenous Sufu from PC3 (B) and DAOY cells (C) transfected with either an empty vector (EV) or Myc-tagged Fbxl17. Non-specific rabbit immunoglobulin G (IgG) was used as a negative control. Detection of light chains of immunoglobulin (IgG) was used to assess the amount of IgG used for each immunoprecipitation reaction. Treatment with MLN4924 (2 M) was started 5 hours before cell collection.


D . of Fbxl17 mRNA in PC3 DAOY transfected with a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) ( 2 ) . were serum starved for 24 hours in serum - reduced medium treated with SAG ( 100 nM ) for 24 hours prior collection .

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D. Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2). Cells were serum starved for 24 hours in serum-reduced medium and treated with SAG (100 nM) for 24 hours prior collection.


E . Sufu in PC3 DAOY treated as in D . GAPDH was used as a loading control . Representative image of three independent experiments is shown .

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E. Sufu protein levels in PC3 and DAOY cells treated as in D. GAPDH was used as a loading control. Representative image of three independent experiments is shown.


F . Sufu in PC3 DAOY infected with an empty backbone retrovirus ( EV ) or a retrovirus expressing Myc - tagged Fbxl17 . Representative image from three independent experiments is shown .

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F. Sufu protein levels in PC3 and DAOY cells infected with an empty backbone retrovirus (EV) or a retrovirus expressing Myc-tagged Fbxl17. Representative image from three independent experiments is shown.


G . Detection of Sufu following cycloheximide ( CHX ) treatment for indicated hours , Fbxl17 depletion using two different siRNAs . ( Hrs = hours ) . Representative image of two independent experiments is

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G. Detection of Sufu protein levels following cycloheximide (CHX) treatment for the indicated hours, and Fbxl17 depletion using two different siRNAs. (Hrs=hours). Representative image of two independent experiments is shown


A . Detection of polyubiquitylated species of endogenous Sufu co - immunopurified from DAOY transfected with Myc - tagged ubiquitin ( Ub ) either in presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) . MG132 ( 10µM ) was added in samples .

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A. Detection of polyubiquitylated species of endogenous Sufu co-immunopurified from DAOY cells transfected with Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


B . Detection of polyubiquitylated species of Sufu upon co - transfection of HEK293T with HA - tagged Sufu Myc - tagged ubiquitin ( Ub ) either in presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) . MG132 ( 10µM ) was added in samples .

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B. Detection of polyubiquitylated species of Sufu upon co-transfection of HEK293T cells with HA-tagged Sufu and Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


C . Fbxl17 mRNA relative in DAOY HEK293T upon Fbxl17 depletion with either a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) ( 2 ) .

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C. Fbxl17 mRNA relative levels in DAOY and HEK293T cells upon Fbxl17 depletion with either a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2).


D . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T co - transfected with HA - tagged Sufu , Myc - ubiquitin ( Ub ) along with either Flag - tagged Fbxl17 wild type ( WT ) or a mutant lacking F - box domain ( Fbxl17ΔF ) . MG132 ( 10µM ) was added in samples .

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D. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.


E . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T co - transfected with Myc - tagged ubiquitin ( Ub ) , Flag - tagged Fbxl17 HA - tagged Sufu WT or Sufu mutant K257R . MG132 ( 10µM ) was added in samples .

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E. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with Myc-tagged ubiquitin (Ub), Flag-tagged Fbxl17 and HA-tagged Sufu WT or Sufu mutant K257R. MG132 (10µM) was added in all samples.


A . Detection of Myc - tagged Fbxl17 after immunoprecipitation of HA - tagged Sufu WT or Sufu S342 / 6A S342 / 6D , as indicated . HEK293T were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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A. Detection of Myc-tagged Fbxl17 after immunoprecipitation of HA-tagged Sufu WT or Sufu S342/6A and S342/6D, as indicated. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


B . Detection of Flag - tagged Gli1 Myc - tagged Fbxl17 binding to immunoprecipitated HA - tagged Sufu Wild - Type ( WT ) or to Sufu S352F . An Empty Vector ( EV ) was used as a negative control . HEK293T were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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B. Detection of Flag-tagged Gli1 and Myc-tagged Fbxl17 binding to immunoprecipitated HA-tagged Sufu Wild-Type (WT) or to Sufu S352F. An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


C . Detection of phosphorylated Sufu on S352 / T353 after immunoprecipitation of HA - tagged Sufu WT Sufu 351 - 353 AAA , as indicated .

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C. Detection of phosphorylated Sufu on S352/T353 after immunoprecipitation of HA-tagged Sufu WT and Sufu 351-353 AAA, as indicated.


D . Detection of Myc - tagged Fbxl17 binding to Sufu full ( FL ) or to following Sufu peptides : Sufu 340 - 360 ( APSRKDSLESDSSTAIIPHEL ) ; Sufu 340 - 360 [ S352P / T353P ] ( phosphorylated on residues S352 T353 ) ; Sufu 351 - 372 ( SSTAIIPHELIRTRQLESVHLK ) .

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D. Detection of Myc-tagged Fbxl17 binding to Sufu full length (FL) or to the following Sufu peptides: Sufu 340-360(APSRKDSLESDSSTAIIPHEL); Sufu 340-360 [S352P/T353P] (phosphorylated on the residues S352 and T353); Sufu 351-372 (SSTAIIPHELIRTRQLESVHLK).


E . Detection of HA - tagged Sufu binding to a Myc - tagged Fbxl17 construct encompassing residues 318 to 701 ( corresponding to F - box motif C - terminus region ) assessed by in vitro binding . Both were synthetized in vitro using a T7 - coupled reticulocyte lysate system .

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E. Detection of HA-tagged Sufu binding to a Myc-tagged Fbxl17 construct encompassing the residues 318 to 701 (corresponding to the F-box motif and C-terminus region) assessed by in vitro binding assay. Both proteins were synthetized in vitro using a T7-coupled reticulocyte lysate system.


F . Detection of ubiquitylated Sufu immunoprecipitated from HEK293T co - transfected with HA - tagged ubiquitin ( Ub ) , Myc - tagged Fbxl17 Flag - tagged Gli1 . MG132 ( 10µM ) was added in samples .

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F. Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged Fbxl17 and Flag-tagged Gli1. MG132 (10µM) was added in all samples.


G . Detection of Flag - tagged Gli1 binding to Myc - tagged Fbxl17 co - immunoprecipitated from HEK293T transfected with a non - targeting siRNA ( Control ) or two siRNA to Sufu ( 1 ) ( 2 ) , as indicated .

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G. Detection of Flag-tagged Gli1 binding to Myc-tagged Fbxl17 co-immunoprecipitated from HEK293T cells transfected with a non-targeting siRNA (Control) or two siRNA to Sufu (1) and (2), as indicated.


A . Sufu in mouse embryonic fibroblasts ( MEFs ) Ptch1 + / + , Ptch1 + / - Ptch1 - / - transfected with a non - targeting siRNA ( Control ) or two siRNAs targeting mouse Fbxl17 ( 1 ) ( 2 ) .

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A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2).


B . of Fbxl17 mRNA in MEFs Ptch1 + / + , Ptch1 + / - Ptch1 - / - transfected as in A .

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B. Quantification of Fbxl17 mRNA levels in MEFs Ptch1+/+, Ptch1+/- and Ptch1-/- transfected as in A.


C . of Gli1 mRNA in MEFs Ptch1 + / + Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005).


D . of Bcl2 mRNA in MEFs Ptch1 + / + Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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D. Analysis of Bcl2 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . Detection of Flag - tagged Gli1 binding to HA - tagged Sufu in MEFs Ptch1 - / - upon expression of Myc - tagged Fbxl17 , or Fbxl17 depletion by two siRNAs . For each condition , 200 µg of whole lysate was immunoprecipitated .

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E. Detection of Flag-tagged Gli1 binding to HA-tagged Sufu in MEFs Ptch1-/- cells upon expression of Myc-tagged Fbxl17, or Fbxl17 depletion by two siRNAs. For each condition, 200 µg of whole cell lysate was immunoprecipitated.


F . Immunostaining of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at N - terminus [ Myc ( N ) - Fbxl17 ] or at C - terminus [ Myc ( C ) - Fbxl17 ] . Scale bars : 20 m .

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F. Immunostaining of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N-terminus [Myc(N)-Fbxl17] or at the C-terminus [Myc(C)-Fbxl17]. Scale bars: 20 m.


G . Detection of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably expressing Myc ( N ) - Fbxl17 ( Fbxl17 tagged at N - terminus ) Myc ( C ) - Fbxl17 ( Fbxl17 tagged at C - terminus ) .

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G. Detection of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably expressing Myc(N)-Fbxl17 (Fbxl17 tagged at the N-terminus) and Myc(C)-Fbxl17 (Fbxl17 tagged at the C-terminus).


H . Detection of HA - tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from extracts from Ptch1 - / - MEFs . Asterisk ( * ) indicates a non specific band . Identification of fractions was performed by Lamin A / C GAPDH detection .

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H. Detection of HA-tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1-/- MEFs. Asterisk (*) indicates a non specific band. Identification of cytoplasmic and nuclear fractions was performed by Lamin A/C and GAPDH detection.


A . proliferation of DAOY upon Fbxl17 depletion using a non - targeting siRNA ( Control ) , two siRNAs against Fbxl17 ( 1 ) ( 2 ) or upon reintroduction of a Myc - tagged Fbxl17 construct in Fbxl17 - depleted DAOY using siRNA Fbxl17 ( 2 ) ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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A. Cell proliferation of DAOY cells upon Fbxl17 depletion using a non-targeting siRNA (Control), two siRNAs against Fbxl17 (1) and (2) or upon reintroduction of a Myc-tagged Fbxl17 construct in Fbxl17-depleted DAOY cells using siRNA Fbxl17 (2) (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


B . of Fbxl17 mRNA in DAOY transfected with a non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) ( mean ± from 3 independent experiments , * * p < 0 . 005 ) .

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B. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2) (mean ± SEM from 3 independent experiments, **p<0.005).


C . Sufu in DAOY treated as in A . Representative image of three independent experiments is shown .

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C. Sufu protein levels in DAOY cells treated as in A. Representative image of three independent experiments is shown.


D . proliferation of DAOY transfected with non - targeting siRNA ( Control ) , siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 Sufu . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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D. Cell proliferation of DAOY cells transfected with non-targeting siRNA (Control), siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . of Fbxl17 mRNA in DAOY treated as in D mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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E. Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments, ***p<0.0005).


F . Sufu in DAOY treated as in D . Representative image of three independent experiments is shown .

F. Sufu protein levels in DAOY cells treated as in D. Representative image of three independent experiments is shown.


A . of Fbxl17 mRNA in DAOY transfected with control shRNA or shRNA targeting Fbxl17 . ( mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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A. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17. (mean ± SEM from 3 independent experiments, ***p<0.0005).


B . Detection of Sufu in DAOY transfected with control shRNA or shRNA Fbxl17 . Representative image of three independent experiments is shown .

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B. Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. Representative image of three independent experiments is shown.


C . of Gli1 mRNA in DAOY transfected with control shRNA or shRNA Fbxl17 . ( mean ± from 3 independent experiments , * * p < 0 . 005 ) .

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C. Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. (mean ± SEM from 3 independent experiments, **p<0.005).


D . T2 - weighted magnetic resonance imaging ( MRI ) showing tumour development in rats injected with DAOY transfected with control shRNA or shRNA against Fbxl17 . One representative image for each condition , at 10 is shown . Scale bar : 5mm . E . Graph showing of T2 hyperintensity ( a surrogate marker of tumourgrowth ) between 4 10 post - tumour induction . were injected with DAOY stably expressing control shRNA or shRNA against Fbxl17 ( 10 , 000 / 1µL ) . ( mean ± ; n = 5 ; two - way ANOVA , followed by unpaired t - , * p < 0 . 05 , * * p < 0 . 01 ) .

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D. T2- weighted magnetic resonance imaging (MRI) showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17. One representative image for each condition, at 10 weeks is shown. Scale bar: 5mm. E. Graph showing area of T2 hyperintensity (a surrogate marker of tumourgrowth) between 4 and 10 weeks post-tumour induction. Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 (10,000 cells/1µL). (mean ± SEM; n = 5; two-way ANOVA, followed by unpaired t-test, *p<0.05, **p<0.01).


F . Representative immunohistochemistry image of Vimentin . Scale bar : 1 mm . G . of tumour growth ( vimentin ) in rats injected with DAOY transfected with either control shRNA or shRNA against Fbxl17 ( mean ± ; n = 5 ; unpaired t - , * p < 0 . 05 ) .

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F. Representative immunohistochemistry image of Vimentin staining. Scale bar: 1 mm.G. Quantification of tumour growth (vimentin staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


H . Representative immunohistochemistry image of Ki67 . Scale bar : 0 . 5 mm / 0 . 1 mm . I . of proliferative index ( Ki67 ) in rats injected with DAOY transfected with either control shRNA or shRNA against Fbxl17 ( mean ± ; n = 5 ; unpaired t - , * p < 0 . 05 ) .

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H. Representative immunohistochemistry image of Ki67 staining. Scale bar: 0.5 mm/0.1 mm.I. Quantification of proliferative index (Ki67 staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


A - C . mRNA expression of Sufu , Gli1 Fbxl17 detected in 285 medulloblastoma samples ( GEO accession : GSE37382 ) , stratified by subtype . Level of significance ( p ) for Kruskal - Wallis ( KW ) rank is p < 10 - 8 . using one - way anova gave comparable results .

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A-C. mRNA expression of Sufu, Gli1 and Fbxl17 detected in 285 medulloblastoma samples (GEO accession: GSE37382), stratified by tumor subtype. Level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8. Analysis using one-way anova gave comparable results.


D . Expression of Gli1 Fbxl17 in 285 is shown colored by subgroup ; level of significance ( p ) for Kruskal - Wallis ( KW ) rank is p < 10 - 8 , Spearman ' s rank correlation ( R = 0 . 5640 ) is indicated .

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D. Expression of Gli1 and Fbxl17 in the 285 patients is shown colored by subgroup; level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8, and Spearman's rank correlation (R=0.5640) is indicated.


E . Detection of HA - tagged Sufu wild type ( WT ) HA - tagged Sufu S352F in NIH3T3 after cycloheximide ( CHX ) treatment . Where indicated , were transfected with a non - targeting siRNA ( Control ) or two siRNA to Fbxl17 ( 1 ) ( 2 ) .

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E. Detection of HA-tagged Sufu wild type (WT) and HA-tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide (CHX) treatment. Where indicated, cells were transfected with a non- targeting siRNA (Control) or two siRNA to Fbxl17 (1) and (2).


F . Assessment of Hh pathway activation using Gli1 - luciferase reporter in MEFs Sufu - / - upon reintroduction of HA - tagged Sufu WT or Sufu mutant S352F using a retroviral system . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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F. Assessment of Hh pathway activation using Gli1-luciferase reporter assay in MEFs Sufu-/- upon reintroduction of HA-tagged Sufu WT or Sufu mutant S352F using a retroviral system. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


T2 - weighed brain MRIs of A ( D1 - 3 ) at 28 months of B at ( D4 - 6 ) at 16 months of , showing global brain atrophy as well as periventricular deep matter hyperintensities .

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T2-weighed brain MRIs of patient A (D1-3) at 28 months of age and patient B at (D4-6) at 16 months of age, showing global brain atrophy as well as periventricular and deep white matter hyperintensities.


Quantifications of HOPS / CORVET subunits in fibroblast lysates ( top ) representative immunoblots ( bottom ) , analyzed as in ( C ) . ( E ) VPS33A , ( F ) VPS11 , ( G ) VPS18 .

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Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18.


Immunoblots ( left ) quantifications normalized to control ( right ) of VPS16 , VPS33A VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control ( empty vector ) .

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Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).


of early endosome marker EEA1 in fibroblasts transduced with control or VPS16 - expressing lentivirus by confocal microscopy . ( A ) Representative confocal . Scale bar , 20 μm . ( B ) of EEA1 - stained number intensities ( ca 80 analyzed for each of n = 3 biological replicates ) .

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Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


of intracellular Transferrin trafficking . Fibroblasts fed with fluorescently labeled Transferrin were co - stained with LysoTracker ( G ) or an antibody against LAMP2 ( H ) . Representative ( left ) of fibroblasts transduced with control , with magnified areas demonstrating co - localized ( arrowheads ) . Scale bars 10 μm or 2 μm ( inserts ) . Quantifications ( right ) of co - localization between Transferrin LysoTracker or LAMP2 , respectively , expressed as Pearson correlation coefficients . Colored horizontal bars indicate median values whiskers 5 95 percentiles ( n = 10 optical sections from 3 independent experiments ) .

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Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).


Confocal of fibroblasts transduced with control or VPS16 - expressing lentivirus labeled with LysoTracker . Scale bar , 20 μm . of LysoTracker - stained number intensities ( ca 80 analyzed for each of n = 3 biological replicates ) .

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Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


Representative immunoblots ( bottom ) summary quantifications ( top ) of of LAMP1 ( C ) LAMP2 ( D ) in fibroblasts , normalized to of actin ( ACTB ) expressed as % of controls ( n = 3 biological replicates ) . of lysosomal Cathepsin D ( CtsD ) processing by immunoblotting . Immunoblot to detect CtsD isoforms ( left ) ( right ) of processing , calculated as of CtsD divided by sum of isoforms ( n = 3 biological replicates ) .

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Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).


of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . ( F ) Representative confocal ( optical sections ) of fibroblasts imaged live under basal or serum - starved conditions . Scale bar , 20 μm .

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Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm.


of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . of autophagosomes , defined as green + red ( G ) autophago - lysosomes , calculated as number of red ( but not green ) divided by total number of . Colored horizontal bars denote median values whiskers 5 95 percentiles ( n = 50 - 113 from a total of 3 independent experiments ; for exact values see Appendix Table S1 ) .

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Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1).


Myelination , as reported using Tg ( mbp : gfp - caax ) transgenic , is significantly reduced in vps16 crispants . yellow box indicates region assessed in D . Scale bar 100 μm . Bar graphs represent data as mean ± . Statistical comparisons by Mann - Whitney U ( n = 6 ) . * * p < 0 . 01 .

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Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.


LysoTracker signal colocalizes with astroglial reporter slc1a2b : citrine does not appear to overlap with pan - neuronal reporter elavl3 : GCaMP5G .

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LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.


b . In vitro activity . Left y - axis : 13C2 - oxalate in mM , right y - axis : 13C - formate in mM , x - axis : time in minutes . SYNB8802 ( blue , circles , solid : oxalate , dotted : formate ) control EcN ( pink , triangle , solid : oxalate , dotted : formate ) . Three biological replicates were run plotted separately .

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b. In vitro activity. Left y-axis: 13C2-oxalate in mM, right y-axis: 13C-formate in mM, x-axis: time in minutes. SYNB8802 (blue, circles, solid line: oxalate, dotted line: formate) The control EcN (pink, triangle, solid line: oxalate, dotted line: formate). Three biological replicates were run and plotted separately.


c . In vitro simulation ( IVS ) . Left y - axis : Rate of oxalate degradation in μmol / h / 109 . X - axis = time in hours . Left X - axis 0 - 4 hours , Right X - axis 6 - 48 h . Three biological replicates were run plotted separately . Data points in orange box represent incubation in simulated gastric fluid ( SGF ) . Data points in blue box represent incubation in simulated intestinal fluid ( SIF ) . Data points in pink box represent incubation in simulated colonic fluid ( SCF ) .

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c. In vitro simulation (IVS). Left y-axis: Rate of oxalate degradation in μmol/h/109 cells. X-axis = time in hours. Left X-axis 0 - 4 hours, Right X-axis 6 - 48 h. Three biological replicates were run and plotted separately. Data points in the orange box represent incubation in simulated gastric fluid (SGF). Data points in the light blue box represent incubation in simulated intestinal fluid (SIF). Data points in the pink box represent incubation in simulated colonic fluid (SCF).


b . Acute mouse study of UOx lowering with SYNB8802 . y - axis shows urinary 13 C2 - oxalate normalized to creatine . x - axis shows two groups , EcN ( control , pink ) vs SYNB8802AbxR ( blue ; n = 15 mice per group ) . dots represent each metabolic cage ( n = 3 mice per metabolic cage ) . Error bars are calculated as . ( unpaired t - with Welch ' s correction , * * * * p < 0 . 0001 )

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b. Acute mouse study of UOx lowering with SYNB8802. The y-axis shows the urinary 13 C2-oxalate normalized to creatine. The x-axis shows the two groups, EcN (control, pink) vs SYNB8802AbxR (light blue; n = 15 mice per group). Individual dots represent each metabolic cage (n = 3 mice per metabolic cage). Error bars are calculated as SEM. (unpaired t-test with Welch's correction, **** p < 0.0001)


d . Nonhuman primate model development study . y - axis shows urinary oxalate normalized to creatine . X - axis shows baseline urinary oxalate after overnight fast , urinary oxalate measured 6 hours post administration of either a spinach preparation ( triangle , pink ) or water ( circle , blue ) ( n = 6 for each group ) . dots represent . Error bars are calculated as . ( Two - way ANOVA followed by Sidak ' s multiple comparison , * * * * p < 0 . 0001 ) .

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d. Nonhuman primate model development study. The y-axis shows urinary oxalate normalized to creatine. X-axis shows baseline urinary oxalate levels after overnight fast, and urinary oxalate measured 6 hours post administration of either a spinach preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).


f . Urinary recovery of oxalate in nonhuman primates . y - axis shows change in urinary oxalate from vehicle control . x - axis shows vehicle ( control , grey ) increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatment groups ) . dots represent normalized urinary oxalate for each . Error bars are calculated as . ( One - way ANOVA followed by Tukey ' s multiple comparison , * p < 0 . 05 )

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f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)


g . Urinary recovery of 13C2 oxalate in nonhuman primates . y - axis shows change in urinary 13C2 - oxalate from vehicle control . x - axis shows vehicle ( control , grey ) increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatmentgroups ) . dots represent normalized urinary 13C2 - oxalate for each . Error bars are calculated as . ( One - way ANOVA followed by Tukey ' s multiple comparison , * * p < 0 . 01 )

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g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)


( B ) Time constants of decay , τdecay , of STC isolated pharmacologically in WT FHM2 KI mice . Top , superimposed representative traces of inward current evoked in an astrocyte ( held at - 90 mV ) by a single stimulation ( indicated by arrow ) in a WT slice , before ( trace a ) after ( trace b ) application of a saturating of GluT inhibitor TFB - TBOA ( TBOA ) . STC was obtained by subtracting residual current remaining in presence of TBOA from total inward current ( trace a - b ) ; decay of STC was best fitted by a single exponential function with τdecay = 6 . 53 ms ( in red ) . bar plot shows average values of τdecay of STC isolated pharmacologically in cortical slices ( n = 13 ) from P22 - 23 WT ( N = 7 ) KI mice ( n = 9 ; N = 3 ) . STC τdecay is 17 % higher in KI compared to WT astrocytes ( unpaired t - : P = 0 . 003 ) . Hereafter , n indicates number of slices N indicates number of mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice.


( C ) τdecay of STC isolated using an exponential waveform approximating average TBOA - insensitive current in WT KI mice . Top trace , average normalized TBOA - insensitive current obtained by pooling normalized TBOA - insensitive currents recorded in 16 WT KI . This current was best fitted by an exponentially rising function ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms ( in green ) . STC was obtained by subtracting from total current elicited in astrocyte ( a : same representative trace as in ( B ) ) exponential function A ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms A equal to maximal current measured in astrocyte at about 60 ms after stimulation ( trace c ) ; decay of STC ( trace a - c ) was best fitted by a single exponential function with τdecay = 6 . 49 ms ( in red ) . bar plot shows average values of τdecay of STC isolated as shown in top panel in cortical slices from P22 - 23 WT ( n = 28 ; N = 11 ) KI mice ( n = 27 ; N = 9 ) . STC τdecay is 21 % higher in KI compared to WT astrocytes ( unpaired t - : P < 0 . 0001 ) .

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(C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).


( A ) Isolation of STC elicited by last of a high - frequency train of action potentials . Left : superimposed representative traces of inward current evoked in an astrocyte ( held at - 90 mV ) by extracellular stimulation with a train of 10 pulses ( trace a : black ) a train of 9 pulses ( trace b : brown ) at 50 Hz in a WT cortical slice . inward current elicited by 10th was obtained by subtracting current elicited by 9 pulses from that elicited by 10 pulses ( trace a - b ) . Right : STC elicited by 10th ( trace a - b - c ) was obtained by subtracting exponential function that simulates TBOA - insensitive current elicited by a single ( trace c , obtained as in Figure 1C ) to 10 - 9 pulses difference current ( trace a - b ) . decay of STC elicited by 10th was best fitted by a single exponential function with τdecay = 8 . 04 ms ( in red ) .

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

(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).


( B ) τdecay of STC elicited by 10th of 50 Hz ( τ10 ( 50 Hz ) , left panel ) 100 Hz ( τ10 ( 100 Hz ) , right panel ) trains in layer 1 astrocytes in acute cortical slices from P22 - 23 WT ( n = 23 ; N = 10 for 50 Hz ; n = 18 ; N = 8 for 100 Hz ) KI ( n = 21 ; N = 9 for 50 Hz n = 14 ; N = 7 for 100 Hz ) mice . STC elicited by 10th was obtained as described in ( A ) . STC τ10 ( 50 Hz ) τ10 ( 100 Hz ) are 30 % 37 % higher in KI compared to WT mice , respectively ( unpaired t - : P < 0 . 0001 in both cases ) .

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(B) τdecay of the STC elicited by the 10th pulse of 50 Hz (τ10 (50 Hz), left panel) and 100 Hz (τ10 (100 Hz), right panel) trains in layer 1 astrocytes in acute cortical slices from P22-23 WT (n = 23; N=10 for 50 Hz; n = 18; N=8 for 100 Hz) and KI (n = 21; N=9 for 50 Hz and n = 14; N=7 for 100 Hz) mice. The STC elicited by the 10th pulse was obtained as described in (A). STC τ10 (50 Hz) and τ10 (100 Hz) are 30% and 37% higher in KI compared to WT mice, respectively (unpaired t-test: P < 0.0001 in both cases).


( A ) Simultaneous visualization of GLT - 1a + ( green ) VGLUT1 + ( red ) in first somatic sensory cortex ( SI ) of a WT a KI mouse ( P35 ) . Arrows point to some GLT - 1a + overlaying with VGLUT1 + ( i . e . , GLT - 1a / VGLUT1 related ) ; framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related ( arrowheads ) . microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left right panels 1 µm for enlarged framed areas . ( B ) of GLT - 1a + overlaying with VGLUT1 in P35 WT KI mice . Left , of GLT - 1a + overlaying with VGLUT1 is comparable in WT KI mice ( data were obtained from 14 18 fields of 20 µm x 20 µm from 2 WT 2 KI mice ( 4 sections / ) , respectively ) ( unpaired t - : P = 0 . 52 ) . Right , of GLT - 1a / VGLUT1 related is 19 % reduced in KI mice ( 169 GLT - 1a + analyzed from 2 mice ) compared to WT ( 174 GLT - 1a + analyzed from 2 mice ) ( Mann - Withney U : P = 0 . 016 ) .

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(A) Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e., GLT-1a/VGLUT1 related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.(B) Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice (4 sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1 related puncta is 19 % reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann-Withney U test: P = 0.016).


( A ) Distribution of GLT - 1agold particles in astrocytic processes contacting asymmetric synapses ( perisynaptic astrocytic processes : PAP ) in SI of 5 WT 5 KI ( 2 , 3 ) mice ( P34 - 35 ) . In cortical PAP of KI mice density ( particles / µm2 ) of total - associated gold particles ( arrowheads ) are reduced compared to WT mice , whereas density of cytoplasmicgold particles ( arrows ) is comparable to that in WT ( cf Table I for numerical values statistics ) .

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(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).


( B ) Distribution of GLT - 1agold particles in excitatory axon terminals forming asymmetric synaptic contact ( AxT ) in SI of P34 - 35 WT KI mice . In cortical AxT of KI WT mice , GLT - 1a density is comparable ( arrowheads : - associated gold particles ; arrows : cytoplasmicgold particles ) ( cf Table I ) .

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

(B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).


Left , superimposed representative traces of normalized inward current evoked in a WT ( black trace ) a KI corticalastrocyte ( blue trace ) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices . inset shows in an expanded time scale portion of traces indicated by dotted . slowly decaying current ( τdecay = 2 . 00 s 3 . 04 s for WT KI trace , respectively ) is largely a [ K + ] e - dependent K + current ( IK ) whose decay kinetics provide a measure of rate of K + clearance by astrocytes ( see text ) . Right , time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22 - 23 WT ( n = 21 ; N = 12 ) KI mice ( n = 20 ; N = 8 ) . IK τdecay is 22 % higher in KI compared to WT astrocytes ( unpaired t - : P = 0 . 001 ) .

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

Left, superimposed representative traces of the normalized inward current evoked in a WT (black trace) and a KI corticalastrocyte (blue trace) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices. The inset shows in an expanded time scale the portion of the traces indicated by the dotted line. The slowly decaying current (τdecay = 2.00 s and 3.04 s for the WT and KI trace, respectively) is largely a [K+]e-dependent K+current (IK) whose decay kinetics provide a measure of the rate of K+ clearance by astrocytes (see text). Right, time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22-23 WT (n = 21; N=12) and KI mice (n = 20; N=8). IK τdecay is 22% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.001).