A , B Co - Immunoprecipitation of INO80 subunits from TA of WT linc - MYH KO mice followed by Western Blot . ( B ) Statistical of Co - IP / Western Blot data . amount of RUVBL1 RUVBL2 in INO80 is not changed after deletion of linc - MYH , while amounts of YY1 , WDR5 TFPT pulled down by INO80 increase ( n = 3 WT / 3 linc - MYH KO ; one - way ANOVA , multiple comparisons by Fishers LSD , * p < 0 . 05 ) . Data are mean ± S . E . M .

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A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.


C - K Proximity ligation assays between INO80 YY1 ( C , D ) , INO80 WDR5 ( F , G ) , INO80 RuvBl2 ( I , J ) in proliferating MuSCs . Red signals indicate close proximity of INO80 - V5 respective interacting . Nuclei were stained using DAPI ( blue ) . Statistical evaluation of proximity ligation assays indicating increased interactions of INO80 - V5 YY1 ( E , n = 3 / 3 , 3 independent wells per , two - students t - , * p < 0 . 05 ) of INO80 - V5 to WDR5 ( H , n = 2 ( WT ) / 2 ( KO ) , 3 independent wells per , two - students t - , * * p < 0 . 01 ) . No increase in proximity was detected between Ino80 - V5 Ruvbl2 ( K , n = 2 ( WT ) / 2 ( KO ) , 3 independent wells per , two - students t - , ns : not significant ) . Specificity of analyzed signals was analyzed using single or single secondary antibodies in PLA assays . No signals were detected for these controls . Data are mean ± S . E . M .

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C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.


( A ) Coomassie stained SDS - PAGE showing purified T . thermophila tubulin .

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(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin.


( B ) A representative cryo - electron micrograph of paclitaxel - stabilized T . thermophila MTs .

(B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs.


( C ) Kymographs obtained by TIRF microscopy showing dynamics of T . thermophila MTs at indicated concentrations in µM .

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(C) Kymographs obtained by TIRF microscopy showing the dynamics of T. thermophila MTs at the indicated concentrations in µM.


( D ) Plot showing time course of T . thermophila growth in presence of CA4 ( 20µM ) or parabulin ( 50µM ; error bars corresponding to standard deviation of triplicate biological measurements ) . Control experiments are done with DMSO alone .

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(D) Plot showing the time course of T. thermophila cell growth in the presence of CA4 (20µM) or parabulin (50µM; error bars corresponding to the standard deviation of triplicate biological measurements). Control experiments are done with DMSO alone.


( A ) Plot showing of T . thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in presence of indicated drugs . Error bars represent mean + / - 95 % CI . For each , data is obtained from at least 2 independent experiments . For growth rates growth lengths , n = 41 ( DMSO ) , n = 4 ( 0 . 04uM parabulin ) , n = 34 ( 0 . 04 uM colchicine ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison ; DMSO vs parabulin P < 0001 ( * * * * ) ; DMSO vs colchicine p = 0 . 40 ( n . s . , not significant ) . ( B ) T . thermophila MT growth rates in presence of indicated drugs ( 40 μM ) . Error bars represent mean + / - 95 % CI ( n = 4 ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison ; DMSO vs parabulin P = 0 . 25 ; DMSO vs colchicine P = 0 . 20 ( n . s . , not significant ) .

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(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant).


( D ) Representative kymographs obtained by TIRF microscopy showing effect of parabulin colchicine on dynamic Tt - MTs .

(D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.


( A ) Evaluation of vertebrate toxicity using MTT which detects metabolic activity proportionate to number of viable in a sample . absorbance measurements for treatment conditions were normalized to null control value which is set to 100 % growth . Sub - confluent human fibroblasts were used to visualize potential compound effects during proliferation . results represent average of 9 readings ( 3 biological replicates with 3 technical replicates of each treatment ) ± standard error of mean .

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(A) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample. The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100% growth. Sub-confluent human fibroblasts were used to visualize potential compound effects during proliferation. The results represent the average of 9 readings (3 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( B ) Replication of GFP - expressing T . gondii tachyzoites in intracellular vacuoles can be followed by live imaging . Since vacuoles containing more than eight tachyzoites are not always easy to score , we tallied vacuoles above eight as being 8 + in . At 36 hours , both null vehicle control samples show most vacuoles harbor eight or more tachyzoites ( three or more doublings ) . Vacuoles in parabulin - treated cultures contain significantly fewer , indicating that replication is slowed . graph shows three biological replicates with values normalized to 100 % for each condition each replicate , error bars represent standard error of mean between experiments .

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(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.


( C ) parabulin EC50 value for T . gondii growth was determined using a plaque to measure a reduction in plaque relative to control conditions . Error bars represent standard error of mean . results represent average of 18 readings ( 6 biological replicates with 3 technical replicates of each treatment ) ± standard error of mean .

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(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( D ) Phase contrast of extracellular cultures . Serial passage of T . gondii in 40 - 80 μM parabulin induces accumulation of aberrant tachyzoite forms . These arise with MT defects that disconnect coordinated division daughter budding .

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(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.


A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - . were fixed after 24 , 48 72 hours imaged after with Hoechst . Green arrowheads indicate examples of prometaphase arrested , red arrowheads indicate examples of dead due to Plk1 downregulation . Phenotypic penetrance is indicated at top of each panel with standard deviation derived from 2 independent experiments . Scale bar , 20 μm .

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A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.


B ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - . were fixed after 72 hours imaged after with Hoechst . C ) of measurements from Fig 2B . Data derived from 3 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov .

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B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.


D ) Solid phase transfection of nontargeting ( scrambled ) , GOLGA2 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - . were fixed after 72 hours , stained with Golga2 antibody ( red ) , Hoechst ( blue ) to mark imaged . Scale bar , 50μm . E ) of experiments in Fig 2D . Data derived from 3 independent experiments . P value ( scrambled vs GOLGA2 ) < 2e - 16 , Mann - Whitney U .

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D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test.


F ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - . were fixed after 72 hours , stained with Ki67 antibody ( red ) , Hoechst ( blue ) to mark imaged . Scale bar , 50μm . G ) of experiments in Fig 2F . Data derived from 3 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U .

F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.


H ) Comparison of phenotypic penetrance with liquid solid phase transfection . RPE - 1 , HEK293T , NCI - H358 NCI - N87 were transfected with scrambled or POLR2A targeting gRNAs in in two different numbers ( 2250 or 20000 / well ) . 5 days post transfection viability were measured by CellTiter - Glo . Boxplots represent values from at least 3 independent experiments containing 3 technical replicates .

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H) Comparison of phenotypic penetrance with liquid and solid phase transfection. RPE-1, HEK293T, NCI-H358 and NCI-N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers (2250 or 20000 cells/well). 5 days post transfection cell viability were measured by CellTiter-Glo. Boxplots represent values from at least 3 independent experiments containing 3 technical replicates.


I ) viability measurements after solid phase transfection targeting POLR2A in a panel of lines . Cas9 expressing lines were transfected with scrambled POLR2A targeting gRNA viability was assessed after 5 days . raw values are background subtracted normalized to mock controls . Results are from at least 3 independent experiments containing 3 technical replicates . For lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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I) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines. Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days. The raw values are background subtracted and normalized to the mock controls. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


A ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting RNP complexes into WT RPE - 1TP53 - / - . were fixed after 72 hours imaged after with Hoechst . Scale bar , 50μm . B ) of experiments in Fig 3A . Data derived from 2 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov .

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A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.


C ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting RNP complexes into WT RPE - 1TP53 - / - . were fixed after 72 hours , stained with Ki67 antibody ( red ) , Hoechst ( blue ) to mark imaged . Scale bar , 50μm . D ) of experiments in Fig 3C . Data derived from 2 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U .

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C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.


E ) Four lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


A ) Viability of NCI - N87 NCI - H358 upon transfection of 45 different gRNAs by solid phase transfection . Cas9 expressing NCI - N87 NCI - H358 were seeded on pre - coated plates . 5 days post transfection , viability in each well were measured by CellTiter - Glo . Values were background subtracted , normalized to scrambled controls plotted against each other . Blue dots represent scrambled controls , whereas yellow dots represent POLR2A gRNAs . Green dots represent genes that affect viability in a dependent manner . Results are representative of 3 independent experiments .

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A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments.


B , C ) Logarithm of odds ( LOD ) scores of cancer associated gene KOs in NCI - N87 NCI - H358 derived from screens with gRNAs ( B ) RNPs ( C ) . Bottom panels show distribution of positive ( POLR2A ) negative ( scrambled ) controls for each experiment . LOD score of 3 is highlighted with a dashed , indicating statistical significance .

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B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.


A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting RNP complexes into human lung fibroblasts ( HLF - 1 ) , lung adenocarcinoma ( LAD - 1 ) or RPE - 1TP53 - / - . 72 hours post transfection were stained with Hoechst imaged . Boxplots represent values from 3 independent experiments containing 3 technical replicates . For lines , P values ( scrambled vs Plk1 ) < 0 . 05 . In right panels , representative for each is shown after RNP transfection targeting PLK1 . Arrowheads indicate that are arrested in prometaphase due to Plk1 downregulation . Scale bars indicate 20μm .

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A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting RNP complexes into human primary lung fibroblasts (HLF-1), primary lung adenocarcinoma cells (LAD-1) or RPE-1TP53 -/- cells. 72 hours post transfection cells were stained with Hoechst and imaged. Boxplots represent values from 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs Plk1) < 0.05. In the right panels, representative images for each cell line is shown after RNP transfection targeting PLK1. Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation. Scale bars indicate 20μm.


B ) Two human lung fibroblasts ( HLF ) three lines derived from suffering from either lung squamous carcinoma ( LSC ) or lung adenocarcinoma ( LAD ) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


( C ) Mass spectra of recombinant HTTex1 23Q after 16 hour of co - incubation with by TBK1 showing complete phosphorylation of S13 S16 . lower panel is a representative western blot of same phosphorylation reaction ( upper panel ) using anti - pT3 ( CHDI - 90001528 - 1 ) , pS13 HTT ( CHDI - 90001039 - 1 ) pS16 ( ZCH11020 - generated in house ) , antibodies ( ab ) after indicated phosphorylation reaction times .

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(C) Mass spectra of recombinant HTTex1 23Q after 16 hour of co-incubation with by TBK1 showing complete phosphorylation of S13 and S16. The lower panel is a representative western blot of the same phosphorylation reaction (upper panel) using anti-pT3 (CHDI-90001528-1), pS13 HTT (CHDI-90001039-1) and pS16 (ZCH11020 -generated in house), antibodies (ab) after the indicated phosphorylation reaction times.


( D ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q 72Q eGFP with indicated kinases for 48 hour in HEK293T .

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(D) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells.


( E ) of fold changes in HTT pS13 pT3 ratios to total HTT compared to empty vector ( EV ) control upon coexpression of indicated kinase from experiments like in D .

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(E) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector (EV) control upon coexpression of the indicated kinase from the experiments like in D.


( A ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP , mutants with S to A substitutions at T3 or S13 / S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase - dead ( KD ) for 48 hour in HEK293T ( additional high molecular HTTex1 HTT pS13 band is possibly due to co - occurrence of phosphorylation at both S13 T3 ) . ( B ) of fold changes in HTT pS13 ( upper panel ) pT3 ( lower panel ) to total HTT compared to those of kinase - dead mutant from experiments like in A ( 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 O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP, mutants with S to A substitutions at T3 or S13/S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase-dead (KD) for 48 hour in HEK293T cells (the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co-occurrence of phosphorylation at both S13 and T3). (B) Quantification of the fold changes in the HTT pS13 (upper panel) and pT3 (lower panel) ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in A (n=3).


( C ) Representative western blot of HTT ( ab - MAB2166 ) HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT N543 55Q with indicated kinase for 24 hour in HEK293T . ( D ) of HTT pS13 using Singulex ( using ab - MW1 as capture CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT N543 55Q with indicated kinase for 24 hour in HEK293T from experiment in C ( representative of 3 repeats ) .

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

(C) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells. (D) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C (representative of 3 repeats).


( E ) Representative western blot of HTT ( ab - MAB2166 ) HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with indicated kinase for 24 hour in HEK293T . ( F ) of HTT pS13 using Singulex ( using ab - MW1 as capture CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with indicated kinase for 24 hour in HEK293T from experiments likein E ( 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells. (F) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E (n=3).


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

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

(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 .

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

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 - .

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

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 - .

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

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 - .

O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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. 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 - .

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

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 O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O

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 .

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

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

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 ) .

O O O O O O 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

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 O O O O O O O O O O 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 .

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

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 - .

O O O O B-MEASURED_VAR I-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

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 .

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

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 - .

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

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 .

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

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 .

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

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 .

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

B, 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.